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UNIVERSITY    OF    CALIFORNIA    PUBLICATIONS 
PATHOLOGY 

Vol.  I,  No.  8,  pp.  87-341  February  16,  1907 


(From  the  Hearst  Laboratory  of  Pathology) 

BY 
ALONZO  EXGLEBEKT  TAYLOR. 


(Dr.  M.  Herzstein  of  San  Francisco  has  established  in  the  University  of 
California  a  Lectureship  devoted  to  the  exposition  of  scientific  subjects 
fundamental  to  medicine.  The  following  publication  comprises  in  a  con- 
densed form  the  lectures  scheduled  for  1904-05,  delivered  in  October,  1904. 
They  include,  however,  literature  references  up  to  December  31,  1905.  The 
publication  has  been  delayed  for  over  a  year,  an  indirect  result  of  the  con- 
flagration of  April  18,  1906.) 

INTRODUCTION. 

These  lectures  represent  an  attempt  at  a  brief  systematic 
consideration  of  the  question  of  fermentation  from  the  double 
point  of  view  of  general  chemistry  and  chemical  biology.  They 
contain,  in  addition  to  a  review  of  the  special  literature  of  the 
topic,  unpublished  researches  on  various  aspects  of  the  subject. 
The  scope  of  the  discussions  is  limited  to  fermentations  of 
importance  to  the  animal  economy.  And  of  these  fermentations 
those  only  shall  be  considered  concerning  which  we  possess  data, 
,  sufficient  in  quantity  and  reliable  in  quality,  that  will  enable 
us  to  rest  the  discussion  upon  a  rigid  objective  basis.  It  must 
be  realized  that  such  an  attempt  will  in  reality  state  the  problem 
rather  than  define  the  extent  of  its  present  solution,  since  the 
data  are  in  truth  scant,  and  many  adventitious  variables  compli- 
cate the  investigations.  It  will  I  believe  be  advantageous  at  the 
outset  to  state  formally  the  conclusions  towards  wrhich  our  theo- 
retical analogies  and  our  experimental  researches  lead.  It  may 
seem  anomalous  to  place  conclusions  in  the  introduction.  This 
is  justified  by  the  fact  that  the  problem  is  not  one  that  has  arisen 
directly  from  the  experimental  investigations  upon  fermenta- 
tions, but  is  one  that  has  been  extended  to  fermentations  as  the 


205459 


88  University  of  California  Publications.       [PATHOLOGY 

result  of  the  modern  development  of  chemical  dynamics.  The 
study  of  fermentation  consists  in  the  attempt  to  verify  there  the 
laws  that  obtain  in  general  chemistry — the  attempt  to  determine 
in  a  complex  biological  material  the  existence  and  validity  of  the 
laws  that  have  been  proved  in  simple  chemical  material.  Under 
these  circumstances  to  state  the  conclusion  is  equivalent  to  stating 
the  problem. 

Fermentations  shall  be  considered  as  accelerations  of  existing 
reactions.  As  accelerations  of  existing  reactions  they  are  ranged 
with  the  reactions  of  catalysis.  In  every  reacting  system  there 
is  a  driving  force  and  a  passive  resistance  to  the  reaction.  The 
catalysor  is  related  only  to  the  condition  of  passive  or  internal 
resistance.  Anything  that  will  lower  this  passive  resistance  will 
accelerate  the  reaction  velocity.  The  positive  catalysor  and  in- 
crease in  temperature  both  act  by  such  a  reduction  in  the  internal 
resistance.  The  modus  operandi  of  catalytic  acceleration  is  in 
general  defined  as  a  succession  of  intermediary  reactions — a  defi- 
nition that  applies  directly  only  to  reactions  in  a  homogeneous 
system.  Fermentations  are  considered  as  limited  and  reversible 
reactions.  There  is  no  known  essential  difference  between  an  in- 
organic positive  catalysor  and  a  ferment;  there  is  no  distinction 
in  dynamics  between  so-called  formed  and  unformed  ferments. 
Cells  induce  fermentations  only  through  the  agency  of  chemical 
substances  elaborated  by  them.  All  fermentations  tend  to  obey 
the  laws  of  chemical  kinetics;  the  experimental  deviations  are 
proportional  to  the  multiplicity  of  adventitious  variables  and  to 
the  difficulty  in  the  definition  of  the  experimental  magnitudes. 
The  greater  the  control  in  the  experiment,  the  closer  the  approxi- 
mation to  the  theoretical  law. 

The  experimental  study  of  a  fermentation  may  be  thus  form- 
ulated in  specific  terms : — 

The  primary  reaction.  This  is  a  problem  of  organic  chem- 
istry. 

Relation  of  mass  of  substrate  to  reaction  velocity. 

Relation  of  mass  of  ferment  to  reaction  velocity. 

Relation  of  concentration  in  entire  system  to  reaction  velocity. 

Relation  of  temperature  to  reaction  velocity.  Temperature 
optimum. 


VOL.  1.]  Taylor. — On  Fermentation.  89 

Reversion  of  reaction  by  ferment  action. 

Relation  of  ferment  to  the  products  of  reaction. 

Auto-acceleration ;  zymo-excitors ;  zymo-depressors. 

Inactivation  of  ferment. 

Secondary  reactions. 

Nature  of  ferment.  Colloidal  properties.  Relation  of  ac- 
tivity to  history  and  method  of  preparation. 

The  control  of  the  conditions — the  purity  of  the  reacting  com- 
ponents and  of  the  ferment,  the  temperature,  concentration,  and 
the  inhibition  or  inactivation  of  the  ferment— will  determine  the 
reliability  of  the  results.  It  is  to  be  confessed  that  too  often  such 
control  has  not  been  attained.  The  more  nearly  the  experiment 
approximates  the  conditions  of  an  ideal  chemical  experiment,  the 
more  credible  the  results.  To  be  entirely  definite  the  results 
should  be  quantitative  and  capable  of  a  mathematical  interpre- 
tation. The  problem  of  fermentations  differs  only  in  degree 
from  the  problem  of  catalytic  reactions,  directly  in  proportion 
to  our  inability  to  study  all  the  aspects  as  above  stated,  to  control 
the  variables  and  to  define  the  experimental  magnitudes.  These 
conditions  have  thus  far  been  best  attained  in  connection  with 
the  study  of  the  ferments  derived  from  the  vegetable  or  lower 
orders  of  animal  life. 

The  statement  that  fermentations  are  to  be  regarded  as  ac- 
celerations of  existing  reaction  demands  a  certain  restriction. 
The  earlier  chemists  in  general  regarded  catalytic  reactions  as 
reactions  de  novo;  the  present  general  currency  of  the  opposite 
proposition  is  due  largely  to  the  influence  of  Ostwald  and  Victor 
Meyer,  though  the  first  theoretical  statement  was  probably  con- 
tained in  the  thermodynamic  considerations  of  van't  Hoff  bear- 
ing upon  the  relations  between  reaction-velocity  and  temperature. 
We  are  now  in  possession  of  a  large  amount  of  experimental  data 
confirmatory  of  this  view.  An  apparent  objection  to  the  con- 
ception of  fermentations  as  accelerated  reactions  is  contained  in 
the  fact  that  a  single  substance  will  yield  different  products 
under  the  influence  of  different  ferments.  The  fact  is  however 
capable  of  another  interpretation,  one  harmonious  to  the  theory, 
that  the  product  represents  a  stage  in  the  reaction,  and  that 
different  ferments  accelerate  to  different  stages.  Secondary  reac- 


90  Unircrxiti/  of  ('dlifontia  Publications.        [PATHOLOGY 

tions  must  also  be  considered.  Recently,  however,  van't  Hoff1 
and  Wegscheider  have  pointed  out  that  in  special  instances  the 
addition  of  a  positive  catalysor  to  a  system  in  a  state  of  chemical 
rest  may  inaugurate  a  reaction ;  and  it  is  possible  that  this  may 
be  of  not  such  infrequent  occurrence  in  the  domain  of  organic 
substances.  More  detailed  reference  shall  be  made  to  this  later. 
For  all  the  known  fermentations  occurring  in  nature  it  is  clear 
that  the  fermentations  represent  accelerations  of  existing  reac- 
tions. These  auto-reactions  progress  as  a  rule  at  ordinary  tem- 
peratures with  extreme  slowness.  In  many  instances  however 
they  have  been  studied  and  measured.  This  is  true  particularly 
for  the  auto-oxidations  occurring  in  metals  and  the  auto-hydro- 
lyses  noted  in  solutions  of  organic  substances.  We  possess  like- 
wise accurate  studies  upon  such  reactions  between  gases.  Indeed 
Victor  Meyer's  studies  upon  the  slow  reaction  between  gaseous 
oxygen  and  hydrogen  at  ordinary  temperature  were  among  the 
earliest  investigations  tending  to  indicate  that  catalyses  are  accel- 
erated reactions,  and  not  reactions  de  novo. 

CHEMICAL  EEACTIONS  OF  FERMENTATIONS. 
Fermentations  are  best  studied  and  classified  on  the  basis  of 
the  reactions  involved,  as  long  ago  suggested  by  Henninger. 
This  plan  is  simple,  can  lead  to  no  confusion,  demands  no  pre- 
conceptions, and  places  the  trend  of  investigations  in  harmon> 
with  the  general  customs  of  the  mother  science.  For  every  chem- 
ical phenomenon  one  may  enquire:  What  is  the  reaction?  and 
also :  How  and  under  what  conditions  does  the  reaction  proceed  ? 
There  are  excellent  illustrations  of  physico-chemical  studies  of 
chemical  processes  of  which  the  reactions  are  entirely  unknown. 
The  researches  of  Arrhenius,  the  measurements  of  the  relations 
between  specific  biological  bodies  and  their  anti-bodies,  is  an 
illustration  in  point.  For  the  ferments,  however,  we  possess  act- 
ually more  information  upon  the  chemical  nature  of  the  reac- 
tions than  upon  the  laws  under  which  they  proceed.  The  great 
difficulty  in  the  past  has  been  that  so  much  of  the  work  has  been 
concerned  neither  with  the  nature  of  the  reaction  nor  with  the 
question  of  the  physico-chemical  relations,  the  studies  have 
been  conducted  along  vital  lines,  so  that  the  results  are  not 


VOL.  l.]  Taylor. — On  Fermentation.  91 

adapted  to  purposes  of  exact  interpretation.  The  study  of  a 
fermentation  ought  to  be  carried  out  from  the  double  point  of 
view  of  organic  and  physical  chemistry;  the  neglect  of  these 
studies  by  the  organic  chemist  and  the  narrow  formal  treatment 
of  the  problem  by  the  physical  chemist  have  been  of  detriment  to 
the  advance  of  our  knowledge. 

The  most  simple  reactions  of  catalysis  are  the  intramolecular 
rearrangements.  Many  such  instances  are  met  with  in  organic 
substances.  Aldehyde  on  standing  passes  into  the  paraldehyde. 
The  transformation  proceeds  more  rapidly  at  higher  tempera- 
ture, and  is  especially  accelerated  by  the  presence  of  acids.  Thus 

for  acetaldehyde  (Turbaba)  : 

A) CH  —  CH3 

CH3.CHO  +  CH:!.CHO  +  CH.,.CHO  <      >  CH.,  —  CH  ^O 

\0 CH  -  CH3 

The  polymerization  into  paraldehyde  may  be  regarded  as 
due  to  the  conversion  of  the  aldehdye  CH3.CH  =  0  into  CH3. 


CH  ,  following  which  three  of  such  groups  join  in  a  ring 

structure  to  form  a  saturated  substance.  The  modus  of  the  action 
of  the  hydrogen  ions  in  the  acceleration  of  the  polymerization 
is  not  known.  The  reaction  is  reversible,  and  tends  to  an  equi- 
librium. 

A  very  illustrative  reaction  of  this  type  is  the  reversible  for- 
mation of  dianthracine  from  anthracine,  discovered  by  Fritsch 
and  recently  studied  by  Luther  and  Weigert.  This  is  a  photo- 
chemic  reaction,  and  tends  to  an  equilibrium.  The  reaction  of 

light  is  reversed  in  the  dark,  and  thus  the  reaction  is  to  be  writ- 
light 

ten  2  C14H10  ~~^    C28H20.     In  all  probability  such  photochemic 

darkness 
reactions  are  very  numerous.     Nernst  has  indeed  advanced  the 

hypothesis  that  the  formation  of  ozone  from  atmospheric  oxygen 
is  such  a  process. 

Acetone  slowly  undergoes  polymerization  into  di-aceton  alco- 
hol (Koelichen).  The  transformation  is  much  accelerated  by 

alkalies. 

2  CH3.CO.CH3  <=>  (CH3.CO.CH3)2. 

/OH 

CH:i.CO.CH:i  -r  CH:!.CO.CH:J  <=>  (CH3)  2C CH2.CO.CH3. 

The  reaction  is  reversible. 


92 


University  of  California  Publications.       [PATHOLOGY 


An  excellent  illustration  for  the  aromatic  series  is  afforded 
by  the  transformation  of  diazo-amido-benzol  into  p-amido-azo- 
benzol.  Acids  accelerate  (Goldschmidt). 


CBH5.N:N.NH.C(iHs 


CBHS.N:N.C«H4.NH2 


NH 

I 

N 

II 

N 


NH2 


An  exceedingly  pretty  illustration  of  a  catalytic  intramole- 
cular rearrangement  is  seen  in  the  isomeric  ketones  C5C1G0 
studies  by  Zincke  and  Kuester. 


Cl  C 

II 

Cl  C 


•  CC12  C1C 

cci,  <=>  ci2c 


CC1 

CC12 


CO 


\co 


The  reaction  is  reversible,  and  no  matter  from  which  ketone 
one  proceeds  an  equilibrium  will  be  established  in  the  system. 
Though  the  sole  difference  lies  in  the  positions  of  the  double 
chlorines,  the  two  isomers  present  melting  points  as  different  as 
31°  and  92°. 

Fermentations  of  this  type  are  not  yet  known. 

In  the  next  group  of  catalyses  one  substance  is  broken  into 
two  or  more  molecules,  or  for  the  reversed  reaction  two  or  more 
molecules  combine  to  form  one  substance.  Some  of  these  reac- 


VOL.  1.]  Taylor.- — On  Fermentation.  93 

tions  are  of  the  simplest  of  association  and  dissociation.     Such 

are: 

Hydrogen  +  oxygen  <=>  water. 
2  H2  +  O2  <=>  2  H2O. 

Hydrogen  +  iodine  <=>  hydriodic  acid. 
H2  +  I2  <=>  2  HI. 

Sulphur  dioxide  +  oxygen  <=]>  sulphur  trioxide. 
2  S02  +  02  <=>  2  SO3. 

These  are  all  greatly  influenced  by  changes  in  temperature, 
are  typically  reversible,  and  are  markedly  accelerated  by  the 
members  of  the  platinum  group.  Another  good  illustration, 
studied  by  van 't  Hoff ,  is  the  following : 

Dibrom-succinic  acid  <— >  bromo-maleic  acid  +  hydrobromic 

acid. 
COOH.CHBr.CHBr.COOH  <=>  COOH.CBr.CH.COOH.  +  HBr. 

Many  fermentations  are  of  this  order. 

d-glucose  <=>  aethyl  alcohol  +  carbon  dioxide. 
CHoOH.(CHOH)4.COH<=>2  CH3.CH2(OH)  +2  CO2. 

Malic  acid  <=>  lactic  acid  +  carbon  dioxide. 
COOH.CH2.CH(OH).COOH.  <=>  CH3.CH(OH).COOH  +  CO2. 

The  acceleration  of  the  reaction  in  the  case  of  d-glucose  may 
be  accomplished  by  alkali  or  platinum,  furnishing  a  good  illus- 
tration of  the  identical  natures  of  catalytic  and  fermentative 
acceleration,  d-glucose  is  subject  to  other  fermentations : 

.  d-glucose  <==>  two  mol.  of  lactic  acid. 
CH2OH.(CHOH)4.COH<=>  2  CH3.CH(OH).COOH. 

d-glucose  <=>  butyric  acid  +  2  carbon  dioxide  -f-  hydrogen. 
CH2OH.(CHOH)4.COH  <=>  CH3.CH2.CH2.COOH  +  2  CO2  +  H2. 

Lactic  acid  is  the  intermediary  product. 

So  far  as  we  know,  these  different  products  of  the  fermenta- 
tion of  d-glucose  are  not  interchangeable,  but  are  specific  to  the 
particular  ferment. 

The  fermentation  of  sinigrin  by  myrosin  is  possibly  the  most 
complicated  of  the  known  reactions  of  this  group. 

Sinigrin  <^==^>  allyl-sulphocyanide  +  d-glucose  +  acid  pot.  sul- 
phate. 

C10HlsNKSAo  <=>  C3H5.SCN  +  C6H12O«  +  KHSO4. 


94  University  of  California  Publications.       [PATHOLOGY 

Especially  noteworthy  is  the  formation  of  an  inorganic  elec- 
trolyte as  one  of  the  products. 

Most  of  the  ordinary  fermentations  involve  four  or  more 
component  molecules.  The  substrate  combines  with  another 
molecule,  and  then  divides  to  form  two  or  more  molecules.  That 
our  point  of  view  begins  with  the  body  to  be  fermented  is  simply 
a  result  of  historical  development  and  of  common  experience 
with  the  phenomenon.  Dynamically  the  two  processes  in  the 
equation  are  of  the  same  dignity.  We  use  the  term  substrate 
to  indicate  the  substance  that  in  the  ordinary  sense  of  the  term 
is  the  main  component  in  the  reaction,  the  substance  to  be  fer- 
mented. The  term  product  is  applied  to  the  substances  that 
result  from  the  reaction.  Dynamically,  what  would  be  the  pro- 
ducts of  the  reaction  in  the  one  direction  are  the  primary  sub- 
stances with  the  reaction  in  the  other  direction.  Similarly  the 
second  body  in  the  ordinarily  primary  reaction,  the  substance 
that  is  added  to  the  substrate,  is  not  in  common  usage  accorded 
the  same  dignity  given  to  the  substrate;  but  dynamically  it  is 
upon  the  same  plane.  For  example :  ester  -f-  water  =  alcohol  -f 
fatty  acid.  We  term  the  ester  the  substrate,  the  alcohol  and 
fatty  acid  the  products.  But  if  we  mix  the  alcohol  and  fatty  acid, 
ester  will  be  formed,  and  under  such  circumstances  the  ester  is 
the  product.  Now  since  both  reactions  are  always  taking  place 
side  by  side  in  the  system,  the  use  of  the  terms  substrate  and 
products  is  simply  a  matter  of  convenience,  based  upon  the  fact 
that  experiments  at  direct  fermentation  are  common,  while  ex- ' 
periments  at  reversion  are  rare.  The  water  of  course  is  as  essen- 
tial a  component  in  the  reaction  as  the  ester,  and  in  the  reversed 
reaction  the  water  is  as  truly  a  product.  Of  these  fermentations 
there  are  three  large  groups:  hydrolyses,  oxidations,  and  re- 
ductions. 

The  hydrolytic  cleavages  are  extremely  common.  In  these 
reactions  water  is  added,  and  then  the  product  of  the  union  is 
divided  into  two  or  more  molecules  of  one  substance,  or  into  two 
or  more  substances.  In  the  reversions  of  these  cleavage  reactions, 
two  or  more  molecules  combine  to  form  a  larger  molecule,  with 
the  extrusion  of  water.  Hydrolytic  cleavage  seems  to  be  uni- 
versal in  the  world  of  organic  substances;  whenever  these  sub- 


VOL.  1.]  Taylor. — On  Fermentation.  95 

stances  are  dissolved  in  water,  slow  hydrolytic  cleavages  are  in- 
augurated. These  auto-hydrolyses  have  been  demonstrated  in 
a  large  number  of  instances.  The  thermodynamic  demonstration 
of  the  nature  of  the  phenomenon  is  contained  in  the  fact  that 
steam  is  a  universal  hydrolysing  agent  for  these  substances,  and 
since  the  reaction  occurs  rapidly  at  high  temperatures  it  must 
occur  to  some  extent  at  low  temperatures.  The  agent  in  the  auto- 
hydrolysis  may  be  confidently  assumed  to  be  the  hydrogen  or 
hydroxyl  ion  of  the  dissociated  water.  A  further  proof  of  the 
occurrence  of  these  auto-hydrolyses  is  contained  in  the  fact  that 
hydrogen  ions  are  general  accelerators  of  these  reactions  with 
these  substances.  In  a  certain  number  of  these  reactions,  rever- 
sions have  been  demonstrated,  both  by  catalytic  and  enzymic 
agents ;  and  the  occurrence  of  such  reversions  under  appropriate 
conditions  is  postulated  for  all. 

Of  these  hydrolyses  many  illustrations  may  be  given.     Thus : 

Cellulose  +  water  <=>  hexose  -f-  hexose. 

n  (C6H1005)n  +  n  H2  O  <=>  n  C«H12C6  +  n  C6H12O6. 

The  hexose  is  d-glucose.  The  same  reaction  is  noted  for 
glycogen.  The  common  bacterial  fermentation  of  cellulose  yields 
no  sugar,  but  only  gases  and  acetic  and  butyric  acid  (Omelian- 
sky),  carbon  dioxide  being  evolved  in  all  cases,  but  otherwise 
either  hydrogen  or  methane.  For  starch  and  inulin  similar 
relations  hold. 

n   (C»H1005)U  -!-  n  H2O  <=>  n  C6H12O6. 

For  starch  the  products  are  d-glucose  when  acids  are  em- 
ployed as  the  catalysor,  maltose  when  diastatic  ferment  is  em- 
ployed: from  inulin  only  laevulose  is  secured.  In  the  acid 
hydrolysis  of  starch  the  process  passes  through  the  stage  of  mal- 
tose and  ends  with  the  formation  of  the  hexose ;  in  the  diastatic 
fermentation  the  process  stops  at  the  stage  of  the  disaccharide. 
In  all  of  these  hydrolyses  of  polysaccharides,  the  reactions  pass 
through  many  substages. 

The  disaccharides  undergo  similar  cleavages,  termed  inver- 
sions. These  follow  the  general  type: 

Disaccharide  +  water  <=>  hexose  +  hexose. 
(C12H22On)  +  H,0  <=>  C6H12Oe  +  C6H1206. 


96  University  of  California  Publications.       [PATHOLOGY 

In  the  case  of  sacchrose  the  products  are  d-glucose  and  d- 
laevulose ;  maltose  yields  only  d-glucose ;  lactose  yields  d-glucose 
and  d-galactose,  and  rafinnose  yields  d-glucose,  d-galactose  and 
d-laevulose.  These  reactions  appear  to  occur  directly,  in  the 
ordinary  sense  of  the  term. 

Closely  related  to  the  inversions  of  the  disaccharides  are  the 
hydrolytic  cleavages  of  the  glucosides.  Glucosides  are  combina- 
tions of  a  hexose,  not  with  another  hexose  as  in  the  case  of  a 
disaccharide,  but  usually  with  an  aromatic  substance,  an  alcohol 
or  an  aldehyde.  Thus  their  hydrolyses  follow  the  type  of  the 
inversions.  The  general  type  may  be  illustrated  by  helicin. 

Heliein  +  water  <^=^>  salicylic  aldehyde  +  d-glucose. 
C13H1007  +  H20  <=>  OH.C6H4.CHO  +  C.HUO.. 

There  is  a  wide  range  of  variety  in  the  second  components  of 
these  compounds.  Thus  arbutin  yields  hydroquinon;  phlorid- 
zin,  phloretin ;  tannin,  gallic  acid ;  gaultherin,  methylsalicylic 
acid;  while  amygdaline  yields,  in  addition  to  d-glucose,  hydro- 
cyanic acid  and  benzoic  aldehyde. 

For  many  of  these  hydrolyses  of  poly-  and  disaccharides  auto- 
hydrolysis  has  been  demonstrated  directly.  For  all  of  these  re- 
actions hydrogen  ions  act  as  positive  catalysors.  For  sonic  of 
them  the  colloidal  metals  of  the  platinum  group  have  been  shown 
to  act  as  accelerators.  Ferments  of  the  cytase  type  accelerate 
the  hydrolysis  of  cellulose,  ferments  of  the  diastase  type  act  posi- 
tively for  the  group  of  polysaccharides,  and  enzymes  of  the  type 
of  invertase  accelerate  the  cleavage  of  the  disaccharides  and  glu- 
cosides. It  is  a  noteworthy  fact  that  the  fermentation  of  poly- 
saccharides, disaccharides  and  glucosides  is  an  act  of  hydrolysis; 
the  fermentations  of  the  hexoses  is  not  an  act  of  hydrolysis. 
There  are  possibly  exceptions  to  this  rule;  thus  there  is  pre- 
sumed to  be  a  direct  fermentation  of  lactose  into  lactic  acid ;  but 
as  a  rule  the  fermentation  of  the  higher  sugars  is  a  hydrolytic 
cleavage,  while  the  fermentation  of  the  primary  sugars  is  a  direct 
intramolecular  cleavage.  Hydroxyl  ions  are  more  prominent  as 
a  catalysor  of  the  reactions  of  the  primary  sugars  than  of  those- 
of  the  poly-saccharides. 


VOL.  l.]  Taylor. — On  Fermentation.  97 

The  cleavages  of  albuminous  substances,  that  we  term  diges- 
tion, are  all  hydrolyses.  The  general  reaction  runs : 

Protein  +  water  <^=^>  amido  acids  +  amido  acids. 

There  are  many  substages  in  the  process.  The  end  products 
comprise  quite  a  number  of  different  amido  acids.  These  hydro- 
lyses are  also  accelerated  by  hydrogen  ions,  and  to  some  extent 
by  colloidal  platinum.  The  auto-hydrolysis  has  been  experi- 
mentally demonstrated  for  several  members  of  the  protein  group. 
Cleavage  with  steam  was  indeed  one  of  the  oldest  methods  em- 
ployed for  obtaining  products  of  protein  hydrolysis.  None  of 
these  reactions  have  been  reversed. 

The  fermentations  of  the  fats  are  likewise  instances  of  hydro- 
lytic  cleavage.  All  esters,  both  the  synthetic  esters  and  the  nat- 
ural fats,  are  hydrolyzed  according  to  the  general  equations : 

Aethyl   acetate  +  water  <=>  a  ethyl  alcohol  +  acetic   acid    (Ost- 

wald,  Wys,  Knoblauch). 
CH3.CO.O.CH3.CH2  +  H2O  <=>  CH3.CH2.OH  +  CH3COOH. 

Olein  triglyceride  +  water  <=>  oleie  acid  +  glycerine. 
C3H3(C18HM0,),  +  3H20  <=>  3  C^A  +  CH2(OH).CH(OH). 
CH2(OH). 

These  reactions  are  typically  and  measurably  reversible.  The 
reversibility  of  the  fat  splitting  enzyme  has  also  been  demon- 
strated for  several  fats.  The  simple  reactions  are  greatly  accel 
erated"  by  hydrogen  ions,  and  to  some  extent  by  the  colloidal 
metals  of  the  platinum  group.  In  many  respects  esters  present 
the  best  opportunities  for  the  study  of  ferment  action. 

It  is  thus  seen  that  the  fermentation  of  the  members  of  the 
three  great  groups  of  foods — protein,  carbohydrate  and  fat — are 
instances  of  the  enzymic  acceleration  of  hydrolytic  cleavages. 
These  hydrolyses  are,  in  the  conditions  in  which  they  occur  in 
nature  as  well  as  under  the  circumstances  of  experiments,  mono- 
molecular  reactions,  at  least  in  so  far  as  the  reaction  in  the  direc 
tion  of  the  right  (the  hydrolysis  of  the  substrate)  is  concerned. 
The  water  of  solution  is  so  much  greater  than  the  water  of  com- 
bination, that  the  mass  of  the  water  in  the  system  is  for  practical 
purposes  constant  during  the  duration  of  the  reaction. 


98  University  of  California  Publications.       [PATHOLOGY 

Other  interesting  instances  of  fermentative  hydrolyses  may 
be  described. 

Urea  +  water  <— >  ammonia  +  carbon  dioxide  (Fawsitt). 
CO(NH2)2  +  H2O  <=>  2  NH3  +  CO2. 

Hippuric  acid  -f-  water  <=>  glycocoll  -f-  benzole  acid   (Schmiede- 

berg). 
CeH5.CO.NH.CHo.COOH  +  H2O  <=>  CH2.OH.COOH  +  C6H6. 

COOH. 

Arginine  +  water  <— >  urea  +  ornithin   (Kossel). 
NH2.C:  (NH)2.(CH2).,.CH.NH2.COOH  +  H2O  <=>  CO 

(NH,),,  +  CH2  (NH2).(CH2)2CH(NH2).COOH. 

These  fermentations  are  accomplished  by  ferments  contained 
in  the  liver.  The  cleavages  may  be  accomplished  as  readily  by 
the  action  of  acids  as  by  ferments.  Interesting  are  the  fermenta- 
tions of  the  salts  of  the  vegetable  acids.  A  good  illustration  is 
afforded  by  calcium  formiate  (Hoppe-Seyler).1 

Calcium  formiate  +  water  <=>  calcium  carbonate  +  carbon 

dioxide  -f  hydrogen. 
HCOO\ 

^Ca  +  H,O  =:  CaCO-  +  CO2  +  2  H... 
HCOO 

The  hydrogen  acts  as  an  anti-catalysor. 

Catalytic  accelerations  of  hydrolyses  are  exceedingly  common, 
not  only  in  natural  substances,  but  also  in  synthetic  substances. 

Thus: 

Mon-chlor-acetic    acid  +  water  <^=~^>  glycolic    acid  +  hydrochloric 

acid  (van't  Hoff).2 
CH~.C1.COOH  +  H.O  <=>  CH2.OH.COOH  +  HC1. 

Under  oxidation  fermentations  we  understand  such  accelera- 
tions in  oxidation  as  occur  under  the  influence  of  the  presence 
of  a  ferment.  The  steps  in  these  oxidations  are  not  well  under- 
stood. It  is  not  even  known  that  oxygen  is  always  added  in 
these  reactions,  since  oxidation  can  be  effected  by  the  withdrawal 
of  hydrogen  as  well  as  by  the  addition  of  oxygen.  Biologists 
have  been  inclined  to  group  the  fermentative  oxidations  under 
two  headings,  direct  and  indirect,  according  to  whether  hydro- 
gen peroxide  acted  as  a  carrier,  or  not.  The  data  are  not  suffi- 
cient to  justify  such  a  distinction.  We  ought  not  to  dogmatize 
upon  the  nature  of  these  reactions,  since  we  are  acquainted  with 


VOL.  1.] 


Taylor. — On  Fermentation. 


99 


so  few.  When  the  knowledge  of  the  auto-oxidation  of  inorganic 
substances  now  being  accumulated  (and  which  has  been  recently 
summarized  from  different  aspects  of  the  subject  in  the  publi- 
cations of  Engler  and  Weissberg  and  of  Kastle  and  Loewenhart) 
is  applied  to  the  study  of  these  fermentations,  we  may  expect 
light  to  break  upon  the  subject.  The  best  known  instances  divide 
themselves  in  two  groups :  those  in  which  a  substance  combines 
with  oxygen  to  form  a  single  product ;  and  those  in  which  water 
is  split  off.  Obviously  the  reversion  of  the  latter  would  consti- 
tute hydrolyses. 

Salicylic  aldehyde  +  oxygen  <=>  salicycle  acid  (Jacquet). 
2  OH.C0H4.CHO  +  O2  <=>  2  OH.C0H4.COOH. 


CHO 


OH 


COOI1 


/    OH 


A  soluble  enzyme  of  mammalian  tissues  accelerates  this  re- 
action. 

Hydroquinone  +  oxygen  <=r>  quinone  -\-  water    (Bertram!).1 
2  OH.C0H4.OH  +  O2  <=>  2  CO.C4H4.CO  +  2  H2O. 


C.OH 


CO 


HC 


HC 


CH 


CH 


HC 


HC 


CH 


CH 


CO 


C.OH 


This  fermentation,  which  is  induced  by  the  juice  of  the  Japa- 
nese lac  tree  (laccase),  and  also  by  mammalian  intestinal  secre- 
tions, comprises,  according  to  the  modern  conception  of  these 
substances,  the  transformation  of  a  true  aromatic  substance  into 
an  alicyclic  substance. 

A  good  illustration  is  furnished  by  the  acetic  acid  fermenta- 
tion of  aethyl  alcohol,  occurring  in  two  stages. 


100  University  of  California  Publications.       [PATHOLOGY 

Aethyl  alcohol  +  oxygen  <^=^>  acetaldehyde  +  water. 
2  CH.3CH2.OH  +  (X  <=>  2  CH3.COH  +  2  H2O. 

acetaldehyde  -f-  oxygen  <=>  acetic  acid. 
2  CH3.COH  +  O2  <=>  2  CH3.COOH. 

According  to  recent  investigations,  oxidation  ferments  are 
very  active  in  the  purin  catabolism.  One  illustration  will  suffice. 
Xanthinoxydase  converts  hypothanthin  first  into  xanthin,  then 
into  uric  acid.  (Spitzer,  Wiener,  Burian). 

Hypoxanthin.  Xanthin.  Uric  acid. 

HN  —  CO  HN  —  CO  HN       CO 

I  I  II  II 
HC        C.NH^  — >       OC        C.NH  — >       OC        C.NH 

II  II       ^CH  |         i|          ^CH  |         ||         ^CO 
N  —  C.N^  HN  -  C.N^  HN       C.NH 

Two  interesting  fermentations  are  brought  about  by  the  bac. 
xylinum  (Bertrand).2 

Sorbite  +  oxygen  <^=~^>  sorbose  +  water. 
2  C0H1406  +  02  <=>  2  CeH^Os  +  2  H2O. 

Glycerine  +  oxygen  <=>  di-oxy-acetone  +  water. 
2  CH2(OH).CH    (OH).CH2(OH)  +  O2  <=>  2  CH2(OH).CO.CIL 
(OH)  -(-  2  H2O. 

These  reactions  have  not  been  reversed.  For  all  of  them  in- 
organic accelerators  are  known. 

While  inorganic  accelerators  of  the  oxidation  of  the  inorganic 
salts  of  metals  are  numerous,  fermentative  acceleration  of  such 
oxidations  are  known  certainly  to  exist  only  for  the  nitrites.  The 
reaction : 

Metal  —  NO2  +  O  <=>  Metal  —  NO3 

is  a  reversible  reaction  that  is  going  on  constantly  in  soils  and 
waters  under  the  influence  of  bacteria,  certain  microorganisms 
being  especially  active  in  the  one  or  the  other  direction.  Whether 
in  the  reduction  hydrogen  is  added  and  then  water  extruded  is 
not  known ;  indeed  the  mechanisms  of  the  reactions  have  not  been 
determined  on  account  of  the  complexity  of  the  conditions  under 
which  the  phenomena  occur.  The  oxidation  of  nitrite  to  nitrate 
has  been  long  known  to  agriculture;  the  reduction  of  nitrate  to 
nitrite  has  been  learned  more  recently.  Analogous  fermentations 
have  been  described  for  the  salts  of  sulphur,  but  the  experiments 
lack  confirmation. 


VOL.  1.]  Taylor. — On  Fermentation.  101 

Closely  related  to  the  simple  oxidations  are  the  reactions  with 
hydrogen  peroxide.  Hydrogen  peroxide  tends  to  a  slow  reduc- 
tion, and  the  oxidations  of  substances  by  hydrogen  peroxide  are 
in  general  to  be  regarded  as  accelerations  of  this  auto-reduction. 

Hydriodic  acid  +  hydrogen  peroxide  <^=>>  iodine  +  water 
(Erode). 

2  HI  +  H2O2  <=>  I2  +  2  H2O. 

Sulphurous  acid  -f  hydrogen  peroxide  <=>  sulphuric  acid  + 

water. 
JLS03  +  H,02  <=>  H2S04  +  H20. 

The  oxidation  of  formic  aldehyde  occurs  in  two  stages : 

Formaldehyde  -f-  hydrogen  peroxide  <=]>  formic  acid  +  water. 
H.COH  +  H2O2  <=>  H.COOH  +  H2O- 

Formic  acid  -f  hydrogen  peroxide  <=>  carbon  dioxide  +  water. 
H.COOH  +  H2O2  <=>  CO,  +  2  H2O. 

There  is  some  evidence  tending  to  indicate  that  the  reversion 
of  these  reactions  represents  the  initial  steps  whereby  carbohy- 
drates are  formed  by  plants,  though  the  reactions  are  usually 
written  with  oxygen  alone  (in  the  form  proposed  by  Baeyer  and 
Erlenmeyer)  :  C02  +  H2O  =  H.COOH  +  0  and  H.COOH  =  H. 
COH  +  0.  The  theory  assumes  the  presence  in  the  chlorofyl- 
containing  cell  of  some  enzyme  accelerating  the  reductions,  sub- 
ject to  the  influence  of  the  light.  Since  carbon  dioxide  and  water 
are  universally  present  in  the  atmosphere,  one  has  but  to  assume 
the  removal  or  combination  of  the  formaldehyde  (i.e.,  its  con- 
densation into  sugar)  in  order  to  possess  a  firm  physico-chemical 
basis  for  the  continued  reduction.  The  influence  of  light  has 
recently  been  interpreted  by  Euler1  to  lie  in  a  translocation  of 
the  station  of  equilibrium  in  the  direction  of  the  reaction  of 
reduction.  Loeb1  believes  his  studies  indicate  that  in  the  reduc- 
tion of  carbon  dioxide  to  formic  acid,  carbon  monoxide  is  formed, 
and  that  both  ozone  and  hydrogen  peroxide  appear.  He  suggests 
the  following  reactions : 

2  CO2  <=>  2  CO  +  02. 
CO  +  H20  <=>  H.COOH. 

3  02  <=>  2  03. 

O:!  +  H2O  <=>  H2O2  +  O2. 

Following  which  we  would  have 

CO,  +  H,O2  <=>  H.CHO  +  O3. 


102  University  of  California  Publications.       [PATHOLOGY 

From  formaldehyde  sugar  would  be  formed  by  condensation. 
The  steps  in  the  earlier  stages  of  the  condensation  of  formalde- 
hyde are  not  known ;  it  is  only  certain  that  amido  acids  are  not 
concerned  (Euler).2  This  general  conception  has  been  given  a 
certain  experimental  foundation  in  the  investigations  of  Loeb2  on 
the  effects  of  the  silent  electrical  discharge  in  a  system  contain- 
ing carbon  dioxide  and  water ;  reduction  products  were  obtained 
that  conform  quite  closely  to  the  above  scheme. 

Though  a  certain  amount  of  experimental  work  lies  at  the 
basis  of  these  theories,  it  is  apparent  that  the  analytical  demon- 
strations of  traces  of  hydrogen  peroxide  and  formaldehyde,  ap- 
pearing as  transient  stages  in  a  reaction,  must  be  a  hazardous 
test.  A  further  biological  difficulty  lies  in  the  great  toxicity  of 
formaldehyde,  though  this  could  be  obviated  by  the  assumption 
of  immediate  combination  or  elaboration  into  higher  carbohy- 
drates. 

The  reactions  of  hydrogen  peroxide  are  susceptible  of  accel- 
eration by  a  large  number  of  inorganic  substances,  especially 
by  ferrous  salts,  colloidal  metals  of  the  platinum  group,  molybdic 
and  tungstic  acids.  The  acceleration  of  the  reactions  of  formalde- 
hyde and  formic  acid  may  be  accomplished  with  plant  extracts 
Bach  has  described  the  acceleration  of  the  reduction  of  carbon 
dioxide  to  formic  acid  by  uranium  salts,  a  statement  that  Euler 
was  not  able  to  confirm. 

Fermentative  reductions,  that  is,  the  acceleration  of  reactions 
of  reduction,  have  not  been  long  known.  Indeed  Hoppe-Seyler 
was  inclined  to  deny  the  existence  of  such  fermentations.  Accel- 
erations by  inorganic  catalysors  are  extremely  common.  Cu- 
riously enough,  our  present  knowledge  includes  few  instances  of 
the  fermentative  acceleration  of  reductions  of  organic  substances, 
most  of  the  recognized  reactions  concerning  metallic  salts.  The 
reduction  of  the  nitrate  to  the  nitrite,  mentioned  in  the  previous 
paragraphs,  is  probably  the  most  widely  occurring  reaction  of 
the  type  known.  This  fermentation  occurs  also  in  the  mamma- 
lian juices  (Abelous).  Very  interesting  are  the  reductions  of 
the  acids  and  salts  of  selenium  and  tellurium  by  certain  bacteria 
described  by  Gosio.  These  reductions  follow  the  regular  types : 


VOL.  1.] 


Taylor. — On  Fermentation. 


103 


Selenous  acid  +  sulphurous  acid  <=>  sulphuric  acid  +  selenium 

+  water. 
H2SeO:;  +  2  H2SOa  <=>  2  H2SO4  +  Se  +  H2O. 

Tellurous    acid  -f-  sulphurous     acid  <=>  sulphuric     acid  +  tellu- 
rium +  water. 
H2TeO3  +  2  H2SO3  <=>  2  H2SO4  +  Te  +  H2O. 

We  do  not  know  what  the  reducing  body  that  reacts  with  the 
selenous  and  tellurous  acids  (replacing  the  sulphurous  acid  in 
the  written  reactions)  in  these  bacterial  experiments  actually  is. 
but  the  acceleration  is  very  pronounced,  and  as  stated  is  ob- 
served with  the  -ous  and  -ic  acids  and  their  salts  alike.  In  some 
instances,  the  reduction  may  be  only  to  a  lower  oxide. 

Similar  reactions  occur  with  arsenic,  both  in  the  arsenous  and 
arsenic  states. 

Arsenic  trioxide  +  hydrogen  <=>  arsenuretted  hydrogen  + 

oxygen. 
2  AsO3  +  3  H2  <=>  2  AsH,  +  3  (X 

Arsenic    trioxide  -f-  potassium    acetate  <=>  kakodyl  +  pot.    car- 
bonate +  carbon  dioxide. 
As,03  +  4  KC2H:102  <=>  (As(CH3)2)20  +  2  K2CO3  +  2  CO2. 

These  accelerations  are  produced  in  the  culture  media  of  cer- 
tain bacteria  -.  the  steps  and  details  in  the  reactions  are  not 
worked  out. 

Of  the  fermentative  reductions  of  organic  substances  we  will 
cite  two: 

Xitrobenzol  +  hydrogen  <=^>  anilin.  -\-  water    (Abelous   and  Ge- 
rard). 


C,,H5.NO2  +  3  Ho 

No, 


C,H6.NHa  +  2H20 
NH, 


This  reduction  is  accelerated  by  some  substance  contained  in 
extracts  of  mammalian  tissues  and  in  extract  of  yeast.     What 


104  University  of  California  Publications.       [PATHOLOGY 

the  actual  reducing  component  in  the  reaction  is,  we  do  not 
know ;  it  is  certainly  not  hydrogen  itself. 

Aspartic  acid  +  hydrogen  <=>  ammonium  succinate  (Hoppe- 

Seyler).2 
COOH.CH(NH2).CH2.COOH  +  H,  <=>  COOH.CH2.CH,. 

COOH.NH,. 

This  reduction  is  accomplished  by  many  of  the  common  bac- 
teria, such  as  the  Bacillus  coli  communis.  The  component  sub- 
stance that  reacts  with  the  amido-succinic  acid  is  not  known. 

Other  ferments  are  known  that  do  not  fit  naturally  into  any 
of  these  groups.  An  illustration  may  be  given  in  the  desamida- 
tion  ferments,  substances  that  accelerate  the  replacement  of  the 
amido  group  by  an  hydrolyl  group  in  the  various  amido  acids 
that  are  products  of  the  hydrolysis  of  protein,  and  also  the  re- 
placement of  the  amido  group  in  guanine  and  adenine  by  hydro- 
gen. Thus  the  action  of  guanase  (Jones)  may  be  illustrated  as 
follows : 

Guanine  +  H2  =  hypoxanthin  +  NH3 

HN  —  CO  HN  —  CO 

II  II 

NH2.C        C.NH  s  — >      HC        C.NH^ 

N  —  C.N  -"  N  —  C.N^ 

These  conversions  are  in  the  broad  sense  oxidations,  just  as 
the  removal  of  a  C02  group  constitutes  a  reduction. 

These  illustrations  will  suffice  to  afford  a  cursory  view  of  the 
final  relations  determined  by  the  reactions  of  fermentation  of 
different  types. 

For  nearly  all  of  these  accelerated  reactions  the  slow  auto- 
reactions  are  known.  For  many  of  these  reactions  reversions 
have  been  accomplished.  For  many  of  these  reactions  the  influ- 
ence of  increases  in  temperature  is  known.  These  facts  afford  a 
natural  presumption  that  the  reactions  of  catalysis  and  fermen- 
tation are  essentially  identical. 

In  looking  over  these  reactions  one  cannot  fail  to  be  im- 
pressed with  certain  general  features.  The  heat  relations  in 
fermentations  vary.  In  the  common  hydrolyses  the  products 
have  approximately  the  same  heat  values  as  the  substrate.  The 
oxidations  on  the  contrary  are  exothermic,  the  reductions  endo- 


VOL.  l.]  Taylor. — On  fermentation.  105 

thermic  reactions.  Natural  fermentations  convert  complex  sub- 
stances into  simple  substances,  they  convert  colloids  into  electro- 
lytes, electrolytes  into  simple  gases  and  elements.  Most  of  the 
substances  that  are  commonly  the  substrates  of  fermentations 
are  but  slightly  soluble  in  water,  or  even  incapable  of  true  solu- 
tion but  only  of  colloidal  suspension.  Starches,  cellulose,  pro- 
teins, glucosides,  fats,  all  form  in  water  more  or  less  colloidal 
suspensions,  and  display  further  the  tendency  to  form  hydrogels. 
When  these  substances  are  fermented,  the  products  are  substances 
of  greater  solubility,  quite  devoid  of  colloidal  characteristics, 
with  no  tendency  to  the  formation  of  hydrogels.  Through  the 
fermentation  the  system  has  been  converted  from  a  heterogeneous 
to  a  homogeneous  state.  The  substrates  of  natural  fermentations 
are  usually  substances  of  very  large  molecular  weight,  the  pro- 
ducts possess  small  molecular  weight.  These  substrates  are  al- 
most devoid  of  the  power  of  diffusion,  the  products  diffuse 
readily  as  a  rule.  The  natural  substrates  exert  but  little  depres- 
sion of  the  freezing  point  of  solutions,  the  products  exert  a 
marked  depression  as  a  rule ;  the  substrates  possess  little  osmotic 
pressure,  the  products  marked  osmotic  pressure.  The  substrates 
are  rarely  crystalline,  the  products  are  usually  crystalline.  The 
substrates  are  substances  that  are  not  subject  to  electrolytic  dis- 
sociation. The  products  exhibit  this  property.  The  substrates 
are  substances  with  little  tendency  to  chemical  reaction  as  com- 
pared to  the  products.  There  are  of  course  exceptions  to  these 
statements ;  for  instance,  the  properties  of  the  higher  fatty  acids 
are  in  these  regards  but  little  different  from  their  fats.  The 
natural  fermentations  usually  comprise  the  disintegration  of 
complex  substances  that  have  been  synthesized  in  the  vegetable 
or  animal  organism.  Since  all  fermentations  are  to  be  regarded 
as  reversible  processes,  how  obvious  is  the  suggestion,  first  clearly 
formulated  by  van't  Hoff,  that  the  natural  fermentations  are 
simply  the  reversions  of  the  reactions  whereby  these  substances 
were  formed.  As  we  shall  see,  there  are  no  thermodynamic 
reasons  that  plead  against  this  proposition.  The  fact  that  fer- 
mentations of  the  natural  order  are  exothermic  reactions  has 
been  employed  by  some  biologists  in  support  of  a  teleological 
interpretation  of  the  circumstances,  according  to  which  a  syn- 


10()  University  of  California  Publications.       [PATHOLOGY 

thesis  could  not  be  held  to  be  accomplished  by  the  action  of  a 
ferment.  As  shall  be  pointed  out,  this  interpretation  is  lacking 
in  theoretical  validity. 

It  must  be  conceded  that  the  pendulum  may  swing  too  far 
in  the  opposite  direction,  and  that  fermentations  may  be  ac- 
corded a  too  general  scope  in  physiological  and  pathological  pro- 
cesses.   The  theoretical  possibility  for  such  an  over-generalization 
lies  in  the  very  definition  of  fermentation.     We  have  defined 
fermentation  as  the  acceleration  of  some  existing  reaction  by  a 
substance  formed  in  a  vegetable  or  animal  organism.    When  we 
reflect  further  that  theoretically  every  reaction  is  capable  of  ac- 
celeration, the  boundless  application  of  the  principle  becomes 
apparent.     It  is,  however,  clear  that  chemical  biology  is  in  no 
greater  danger  of  becoming  simply  a  treatise  on  fermentations, 
than  are  inorganic  and  organic  chemistry  in  danger  of  becoming 
reduced  to  the  boundaries  of  catalysis,  since  the  same  principles 
apply  to  each.    In  each  individual  instance  the  question  whether 
a  particular  phenomenon  may  be  a  fermentation  (or  a  catalysis) 
is  plainly  a. matter  of  concrete  demonstration.    And  it  is  because 
this  concrete  demonstration  must  be  so  much  more  difficult  in 
biological  than  in  chemical  questions,  that  there  will  be  a  ten- 
dency  among  biologists  to  overwork  the   principle.     Standing 
bewildered  before  the  apparently  hopeless  complexity  of  a  bio- 
logical problem,  it  is  so  easy  to  say  "ferment  action."    This  very 
tendency  compels  us  to  insist  with  the  greatest  objective  strict- 
ness upon  the  precise  demonstration  of  the  occurrence  of  a  fer- 
mentation.   The  safety  of  the  investigator  lies  in  close  adherence 
to  the  laws  of  general  chemistry  connected  with  the  kinetics  of 
reactions.    Not  only  do  these  control  us  in  our  studies,  but  they 
enable  us  to  investigate  in  a  proper  and  adequate  manner  the 
characteristics  of  a  fermentation,  after  its  identity  as  such  has 
been  established.     The  importance  of  the  study  of  fermentations 
has  been  entirely  underestimated  by  the  biological  world.    While 
in  the  groupings  of  systematic  biology  it  may  have  sufficed  to 
know  simply  that  a  particular  phenomenon  was  a  fermenta- 
tion, for  the  real  study  of  the  chemistry  of  the  functions  of  ani- 
mals and  plants,  that  is  simply  the  stating  of  the  problem.     The 
height  of  biological  research  is  the  reproduction  of  an  act  of 


VOL.  l.]  Taylor. — On  Fermentation.  107 

nature.  To  attempt  to  reproduce  the  chemical  functions  of  or- 
ganized bodies,  one  must  study  fermentations  from  the  point  of 
view  of  the  control  of  its  several  variables. 

Within  recent  years  the  biological  relations  of  the  ferments 
have  been  investigated.  That  there  are  natural  protective  influ- 
ences against  the  action  of  ferments  is  known,  and  certain  native 
forms  of  protein,  for  example,  seem  almost  entirely  resistant  to 
the  action  of  proteolytic  ferments.  Artificial  immunity  to  the 
action  of  a  particular  ferment  has  been  described  as  being  estab- 
lished as  the  result  of  the  reaction  of  an  animal  to  injections  of 
the  ferment,  and  the  term  anti-ferment  has  been  given  to  the 
hypothetical  substance  endowed  with  this  property.  Interesting 
as  these  observations  are,  so  far  as  known  they  do  not  bear  any 
relations  to  the  problem  of  ferment  action,  and  we  shall  give 
them  no  further  consideration. 

APPLICATION    OF   LAWS    OF    CATALYSIS    TO    FEEMENTATIONS. 

We  define  a  catalysis  as  an  acceleration  of  an  already  exist- 
ing reaction  through  the  presence  of  another  body  that  does  not 
appear  in  the  end-products  of  the  reaction.  In  the  specific  in- 
stance there  are  two  criteria  of  a  catalysis.  Every  alteration  in 
velocity  not  dependent  upon  alteration  in  concentration  or  of 
temperature  indicates  catalysis ;  and  in  the  catalytic  acceleration 
there  are  no  stoichiometric  relations  between  the  catalysor  and 
substrate  .or  products.  There  is  theoretically  a  catalysor  for 
every  reaction,  and  every  substance  may  act  as  a  catalysor.  Cer- 
tain classes  of  bodies  possess  to  a  high  degree  this  quality  of 
acceleration :  the  platinum  group,  all  colloidal  metals,  hydrogen 
and  hydroyl  ions,  and  the  oxides  and  oxyhydrates  of  the  ele- 
ments of  varying  valency,  such  as  iron,  manganese,  and  nitrogen. 
The  relations  of  energy  involved  in  a  reaction  are  not  disturbed 
by  a  catalytic  acceleration;  the  result  is  achieved  by  a  diminu- 
tion of  the  chemical  resistance.  The  existence  of  a  large  class  of 
compounds  in  the  natural  state  is  dependent  absolutely  upon 
their  chemical  resistance,  and  were  this  materially  diminshed, 
these  compounds  would  cease  to  exist.  Chemical  resistance  di- 
minishes with  increasing  temperature,  and  there  is  for  each  con- 
centration of  a  chemical  system  an  optimal  temperature.  Cor- 


108  University  of  California  Publications.       [PATHOLOGY 

responding  to  the  physical  state  of  the  system,  we  distinguish 
between  catalyses  in  homogeneous  and  in  heterogeneous  systems. 
The  current  theory  of  physical  states,  well  formulated  by  Spring, 
is  that  from  the  homogeneous  to  the  heterogeneous  state  is  a 
gradual  transition.  In  fermentations  we  deal  with  bodies  that 
present  with  water  less  of  homogeneity  than  solutions  of  pure 
crystalloids,  and  usually  less  of  heterogeneity  than  the  typical 
colloids.  We  shall  see  that  the  behavior  of  fermentations  con- 
firms this  interpretation. 

In  our  consideration  of  the  kinetics  of  catalysis  we  are  con- 
cerned in  the  first  instance  with  the  law  of  mass  action  and 
especially  as  applied  to  a  reaction  of  which  the  products  reunite 
to  form  the  original  substance  to  a  measurable  degree;  and  sec- 
ondly with  the  relations  observed  when  such  a  reaction  is  accel- 
erated by  the  presence  of  a  positive  catalysor  or  enzyme.  Theo- 
retically all  reactions  are  to  be  looked  upon  as  limited  reactions 
and  likewise  reversible;  but  the  point  of  equilibrium  may  be  so 
slightly  removed  from  the  condition  of  a  complete  reaction  as 
not  to  be  analytically  determinable ;  and  it  is  not  always  possible 
to  fix  the  conditions  favorable  to  a  reversion  so  that  the  theoret- 
ical result  shall  become  experimentally  apparent.  What  is  about 
to  be  enunciated  under  these  headings  is  specifically  valid  only 
for  homogeneous  systems ;  the  relations  involved  when  heteroge- 
neous systems  are  concerned  will  be  considered  later. 

The  fundamental  proposition  concerned  in  the  kinetics  of. 
chemical  reactions,  as  early  formulated  by  Wilhelmy,  is  that 
under  constant  conditions  of  experimentation  the  transformation 
in  the  unit  of  time  is  proportional  to  the  mass  of  the  reacting 
bodies.  This  proposition  is  directly  analogous  to  the  Newtonian 
law  for  the  radiation  of  heat  from  a  warm  to  a  cool  body;  the 
radiation  in  time  is  proportional  to  the  difference  in  tempera- 
ture between  the  two  bodies.  The  formulation  may  be  expressed 
in  another  way  in  the  statement  that  whenever  a  transformation 
is  taking  place,  the  rapidity  of  that  transformation  in  a  partic- 
ular moment  is  proportional  to  the  distance  between  the  end- 
point  (the  equilibrium)  and  the  point  in  the  course  of  the  reac- 
tion attained  at  that  particular  moment.  Applied  to  the  con- 
crete instance,  say  to  the  inversion  of  cane  sugar,  we  mean  that 


VOL.  l.]  Taylor. — On  Fermentation.  .109 

in  each  particular  moment  the  amount  of  sugar  inverted  is  pro- 
portional to  the  amount  of  uninverted  sugar  present  in  that 
same  moment.  This  applies  as  stated  only  to  monomolecular 
reactions,  reactions  in  which  the  mass  of  only  one  substance  is 
affected  during  the  reaction.  But  the  general  proposition  applies 
to  bimolecular  and  trimolecular  reactions,  with  the  difference 
that  the  degree  of  reaction  in  time  depends  upon  the  concentra- 
tion of  the  two  or  three  active  masses,  instead  of  depending 
simply  upon  one.  Now  mathematically  all  the  fermentations  oc- 
curring in  the  biological  world  are  monomolecular  reactions,  be- 
cause the  second  body  concerned,  as  water  or  oxygen,  is  present 
in  such  excess  that  alteration  in  its  mass  is  of  almost  no  conse- 
quence. If,  for  instance,  in  the  reaction  cane  sugar  -f-  water  = 
glucose  -f-  fructose  the  amount  of  water  present  \vere  approx- 
imate to  the  amount  required  in  the  reaction,  that  reaction  would 
be  treated  as  a  bimolecular  reaction;  but  as  the  experiment  is 
carried  out  in  dilute  solution,  where  the  water,  as  solvent,  is 
present  in  a  thousand  times  the  amount  required  for  the  water 
as  reagent,  the  reaction  is  equivalent  to  a  monomolecular  reac- 
tion. For  this  reason  we  will  confine  ourselves  to  the  kinetics  of 
the  monomolecular  reaction. 

This  proposition  is  expressed  in  the  general  differential  equa- 
tion —  ^  =  C  (A — x) .  A  is  the  original  amount  of  substrate,  x 
the  amount  of  substance  converted  in  the  time  t,  C  is  a  constant. 
The  equation  holds  only  when  the  temperature  and  volume  are 
held  constant,  and  the  system  is  in  a  state  of  certain  dilution. 
When  integrated  and  reduced  to  its  simplest  relations  under  the 
stipulation  that  when  t  =  0  x  also  =  0,  the  equation  becomes 
C~  .log—  — .  The  constant  expresses  the  work  done  under 

£  J3_ J 

constant  conditions.  If,  for  example,  we  determine  with 
a  particular  strength  of  acid  in  an  inversion  experiment  that 
0  =  0.002,  that  means  that  under  constant  conditions  of  tem- 
perature, volume  and  concentration  of  acid,  in  a  solution  of 
sugar  of  the  strength  of  a  gramme-molecule  in  the  liter.  0.002 
gramme-mol.  of  sugar  will  be  inverted  in  the  first  minute,  and 
if  we  could  add  to  the  system  in  each  minute  without  changing 
the  volume  0.002  gramme-mol.  of  sugar,  that  quantity  would  be 
inverted  regularly  each  minute. 


110  University  of  California  Publications.       [PATHOLOGY 

This  simple  relation  becomes  more  complicated  when  we  deal 
with  a  reaction  of  active  and  measurable  reversibility.  Under 
these  circumstances  an  equilibrium  is  established  in  the  system 
when  the  opposing  reactions  just  compensate  each  other.  The 
reaction  in  the  direction  of  the  right  becomes  less  each  minute 
as  the  mass  of  substrate  becomes  less ;  the  reaction  in  the  direction 
of  the  left  becomes  greater  each  moment  as  the  mass  of  the  pro- 
ducts of  the  other  reaction  increase.  At  a  certain  point  these 
will  be  equal,  and  from  this  time  no  apparent  change  will  be 
observable  in  the  system.  But  it  must  not  be  supposed  that  the 
reactions  have  stopped ; .  they  continue  as  before  proportional 
to  the  active  masses  of  the  respective  components,  but  since  thev 
are  balanced,  the  system  is  in  equilibrium. 

The  reaction  may  be  written  in  the  following  manner,  using 
as  an  illustration  the  reaction  ester  -|-  water  =  alcohol  -f-  fatty 
acid. 

Ester  +  water  <^=>  alcohol  -f-  fatty  acid. 

ester  water  alcohol  fatty  acid 

Const.  Concentrn.          .  Concentrn.  =Const.  Coneentrn.  .  Ooncentrn 

alcohol  fatty  acid 

Concentrn.  .  Concentrn.  Const. — > 

=  COX  ST. 

ester  water         Const.  < — 

Concentrn.  .  Concentrn. 

CONST,  stands  for  the  constant  of  equilibrium. 

When  a  catalysor  is  added  to  systems  representing  each  of 
these  types  of  reaction,  under  conditions  of  controlled  relations 
in  experiments  writh  pure  substances,  nothing  happens  except 
that  the  time  of  the  reactions  is  shortened.  The  accelerated 
reaction  that  naturally  was  practically  a  complete  reaction  re- 
mains practically  a  complete  reaction;  it  is  simply  completed 
more  quickly ;  and  with  different  quantities  of  catalysor  the  dif- 
ferences are  simply  those  of  degrees  of  rapidity.  The  accel- 
erated reaction  of  measurable  reversibility  remains  a  reaction  of 
reversibility  and  the  point  of  equilibrium  is  not  disturbed  by 
the  acceleration ;  the  c-  "_  =  C  is  simply  reached  more  quickly. 
As  I  have  stated  howrever  the  conditions  must  be  controlled.  The 
concentrations  and  volumes  must  be  held  constant,  in  order  that 
the  relations  of  the  active  mass  or  masses  are  maintained:  the 


VoL-  !•]  Taylor. — On  Fermentation.  Ill 

temperature  must  be  held  constant  because  the  point  of  equi- 
librium may  vary  with  the  temperature ;  and  the  products  must 
not  combine  with  the  catalysor,  for  in  this  manner  also  would 
the  constant  of  equilibrium  be  disturbed.  It  is  also  very  impor- 
tant that  pure  substances  be  employed,  since  traces  of  foreign 
bodies  may  exert  a  very  great  disturbance. 

Let  us  now  examine  some  of  the  conditions  and  variations  in 
detail. 

Initial  Concentration  of  the  substrate.  This  is  not  immate- 
rial, and  it  is  not  true  that  at  all  concentrations  of  the  substrate 
the  reaction  in  any  particular  moment  is  proportional  to  the 
active  mass  of  the  substrate.  This  statement  is  true  only  of 
dilute  solutions,  just  as  the  laws  of  electrolytic  dissociation  hold 
true  only  for  dilute  solutions.  In  the  inversion  of  a  dilute  solu- 
tion of  sugar  the  law  of  Wilhelmy  is  obeyed ;  but  in  the  inversion 
of  a  thick  syrup,  no  such  result  is  obtained.  If  one  endeavors, 
in  accordance  with  the  current  tendency,  to  rest  catalytic  reac- 
tions upon  the  theory  of  ionization,  this  fact  becomes  in  a  general 
sense  intelligible.  A  further  bearing  upon  the  disturbing  action 
of  high  concentrations  of  the  substrate  lies  in  the  fact  that  under 
such  circumstances  the  substrate  participates  in  the  role  of  solv- 
ent ;  the  vapor  pressure  of  the  catalysor  may  be  thereby  altered, 
since  under  such  circumstances  the  solvent  will  be  modified. 
Not  only  is  the  velocity  of  a  reaction  dependent  upon  a  proper 
dilution  of  the  substrate ;  the  order  of  a  reaction  is  likewise 
altered  by  excessive  concentration.  In  a  monomolecular  reaction, 
under  conditions  of  proper  concentration,  when  the  system  is 
diluted  the  times  of  equal  proportional  transformation  are  not 
altered;  doubling  the  concentration  doubles  the  velocity,  Under 
similar  circumstances  with  a  bimolecular  reaction,  when  the  sys- 
tem is  diluted  the  times  of  equal  proportional  transformation 
are  inversely  as  the  initial  concentrations ;  doubling  the  concentra- 
tion quadruples  the  reaction  velocity.  Now  under  conditions  of 
high  concentration  these  relations  do  not  hold,  and  it  is  thus 
necessary  to  provide  for  proper  dilution  when  determining  the 
order  of  a  reaction  as  well  as  when  determining  the  velocity. 
The  necessity  for  high  dilution  of  the  substrate  is  most  urgent  in 
the  case  of  substrates  of  ponderous  molecular  weight. 


112  University  of  California  Publications.       [PATHOLOGY 

Concentration  of  the  products.  This  is  likewise  not  immate- 
rial. In  general,  the  higher  the  concentration  of  the  products, 
the  more  energetic  the  process  of  reversion.  But  in  experimental 
catalysis  it  is  sometimes  found  that  when  the  concentration  of 
the  system  is  high  the  resultant  high  concentration  of  the  pro- 
ducts disturbs  the  course  of  the  reaction. 

Influence  of  temperature.  The  acceleration  of  reaction  veloci- 
ties by  increase  in  temperature  holds  good  for  catalytic  reactions 
as  well  as  for  simple  ones.  For  such  work  the  simple  formula  of 

j.  y f 

Arrhenius   will    usually    suffice:    ln-rL  —  A(-   '      2),    A    being 

K2  J.  !   •    J-  2 

the  constant,  and  k±  and  k2  the  velocity  constants  of  the  reac- 
tion at  the  absolute  temperatures  T!  and  T2.  This  equation  has 
been  tested  on  many  catalytic  reactions.  Corresponding  very 
closely  in  its  results  to  the  above  equation  is  the  empirical  rule 
of  van 't  Hoff  that  for  every  ten  degrees  increase  in  temperature 
the  velocity  of  a  reaction  is  doubled,  i.e.,  the  time  required  to  do 

a  unit  of  transformation  is  reduced  one-half, v   /y10=  2+ .  For 

ferments  the  rule  holds  good  in  a  restricted  sense.  The  rule 
holds  for  most  ferments  from  about  15°  to  35°  ;  above  this  the 
increase  in  velocity  is  often  much  more  than  predicated  by  the 
rule,  while  above  45°  there  is  a  rapid  fall  to  zero.  The  fall  is 
the  result  of  the  destruction  of  the  ferment,  a  condition  not  con- 
templated in  the  rule.  As  a  whole  however,  in  consideration  of 
the  complexities  attending  the  experimentation,  the  concordance 
of  the  facts  with  the  theory  is  quite  good. 

A  moment's  consideration  of  the  thermodynamic  relations 
concerned  will  indicate  that  the  formulae  could  not  be  expected 
to  hold  good  unless  the  catalysor  remained  unaltered  and  no 
secondary  reactions  intervened.  These  conditions  are  rarely 
present  in  a  fermentation.  The  best  evidence  of  this  lies  in  the 
experimental  fact  that  the  temperature  optimum  is  a  very  in- 
constant and  shifting  moment,  and  that  the  catalysor  is  inacti- 
vated in  most  fermentations.  The  conditions  surrounding  the 
experiment  have  a  great  deal  to  do  with  the  temperature  opti- 
mum; in  some  work  with  the  digestion  of  protamine  by  trypsin, 
for  example,  I  found  that  the  presence  of  a  little  of  the  vulcan- 
ization powder  from  the  inside  of  new  rubber  tubing  would 
lower  the  temperature  optimum  several  degrees.  The  principal 


VoL-  L]  Taylor. — On  Fermentation.  113 

factor  however  lies  in  the  reactions  between  the  solvent  and  the 
catalysor. 

It  is  a  common  error  to  suppose  that  a  temperature  optimum 
is  peculiar  to  ferment  action.  Every  reaction  has  a  temperature 
optimum,  and  this  optimum  is  usually  altered  (lowered)  in  a 
catalytic  acceleration.  The  system  S02  -(-  0  =  S03  (Knietsch) 
has  its  temperature  optimum  about  900° ;  at  1000°  the  system 
is  in  equilibrium,  the  velocity  of  the  reactions  being  however 
very  slow.  In  the  presence  of  platinum  the  temperature  opti- 
mum is  lowered  to  about  450°,  the  velocity  is  rapid,  and  as  the 
reversed  reaction  (the  dissociation  of  S03)  does  not  occur  to 
any  material  extent  below  500°,  the  acceleration  of  the  reaction 
is  very  effective.  The  catalytic  acceleration  of  the  formation  of 
wrater  from  oxygen  and  hydrogen  also  displays  a  sharp  optimum 
(Ernst).  Ferments  are  notable  simply  for  the  lowness  of  the 
temperature  at  their  optimum,  and  for  the  narrowness  of  the 
optimal  range.  The  latter  condition  has  lost  much  of  its  signifi- 
cance since  we  have  learned  that  the  sharp  descent  of  the  curve 
from  the  optimal  temperature  is  due  in  large  part  to  the  destruc- 
tion of  the  ferment. 

Relations  of  the  catalysor.  The  typical  positive  catalysor 
simply  accelerates  the  velocity  of  a  reaction,  without  altering 
the  nature  of  the  reaction,  the  order  of  the  reaction,  the  char- 
acter or  yield  of  the  products,  the  point  of  equilibrium,  and 
without  being  itself  altered  in  the  process.  It  is  just  as  though 
there  were  a  certain  resistance  to  the  progress  of  the  reaction,  and 
the  presence  of  the  catalysor  diminished  this  resistance.  These 
conditions  are  usually  maintained  in  inorganic  catalyses,  are  as 
a  rule  not  fully  maintained  in  the  acceleration  of  reactions  in- 
volving organic  substances,  while  they  may  be  said  never  to  be 
fully  realized  in  fermentations.  Since  the  presence  of  the  cata- 
lysor simply  accelerates  the  reaction,  it  ought  in  general  to  do  so 
proportionally  to  the  quantity  of  the  catalysor  employed,  and  this 
is  usually  true.  Especially  for  the  catalytic  action  of  hydrogen 
ions  has  this  relationship  been  shown  to  be  true;  the  catalytic 
action  of  acids  is  related  to  their  electrolytic  dissociation.  The 
same  holds  true  for  hydroxylions.  For  other  catalysors  the  same 
rule  has  in  general  been  found  to  hold  good.  There  are  however 


114  University  of  California  Publications.       [PATHOLOGY 

several  well  studied  reactions,  for  which  the  catalytic  accelera- 
tion is  not  proportional  to  the  quantity  of  catalysor.  Thus 
Noyes  has  shown  that  in  the  reaction  between  stannous  chloride 
and  ferric  chloride  the  catalytic  acceleration  of  acids  is  propor- 
tional to  the  square  of  the  hydrogen  ions;  and  in  the  catalytic 
reduction  of  hydrogen  peroxide  by  colloidal  platinum  the  accel- 
eration is  not  proportional  to  the  quantity  of  platinum  (Bredig). 
For  ferments  the  relations  have  not  been  well  studied,  and  most 
of  the  reported  statements  are  not  worthy  of  credence  because  of 
uncontrolled  methods  of  experimentation. 

The  relation  of  the  mass  of  ferment  to  the  acceleration  of 
the  reaction  may  be  usually  expressed  in  the  equation  of  Bredig.1 

f1  W         tm 

7^  =  ( ^  )    •     The  C  is  the  constant  of  velocity  for  the  ferment 

O2  £ 2 

concentration  F,  m  is  a  constant.  For  the  common  catalysors, 
m  =  1.  According  to  Pawlow,  m  is  1/2  for  ferments,  that  is,  the 
so-called  rule  of  Schuetz  holds.  With  the  more  careful  control 
and  measurement  of  ferment  reactions,  however,  m  is  more  and 
more  often  being  determined  to  be  1.  The  proportionality  holds 
for  low  concentrations. 

To  the  statement  that  the  order  of  a  reaction  is  not  altered 
by  the  presence  of  a  positive  catalysor,  exceptions  seem  to  exist. 
The  reaction  H202  -|-  2  HI  =  2  H20  -)-  I2  depends  upon  the  con- 
centration of  the  two  reacting  bodies ;  that  is,  it  is  a  bimolecular 
reaction.  Brode  studied  the  acceleration  of  the  reaction  under 
the  influence  of  molybdic  acid  and  iron  sulphate,  and  found 
that  under  these  circumstances  the  velocity  of  the  reaction  was 
a  function  of  the  concentration  of  hydrogen  peroxide— that  is, 
the  presence  of  the  catalysor  had  converted  the  reaction  from 
one  of  the  second  to  a  reaction  of  the  first  order.  There  exist 
also  other  studies  suggesting  a  similar  shifting  in  the  order  of 
the  reaction,  though  in  none  have  the  relations  been  so  carefully 
worked  out.  The  alteration  is  unquestionably  to  be  explained 
upon  the  basis  of  intermediary  reactions. 

The  statement  that  the  station  of  equilibrium  is  not  trans- 
located by  the  presence  of  a  catalysor  seems  to  hold  absolutely 
good  unless  the  catalysor  is  altered  in  the  progress  of  the  reac- 
tion, and  there  is  for  this  statement  experimental  evidence  as 
well  as  thermodynamic  theory.  The  acceleration  of  the  catalysor 


VoL-  !•]  Taylor.— On  Fermentation.  115 

per  se  simply  multiplies  the  <~  and  ~>  of  the  equation,  the  C 
is  not  aft'ected  by  the  acceleration.  When  however  the  catalysor 
is  altered  during  the  course  of  the  reaction,  it  m^ans  that  the 
substrate,  the  products  or  the  solvent  has  entered  into  reaction 
with  the  catalysor,  and  as  a  consequence  the  concentration  of 
one  or  the  other  member  of  the  system  has  been  altered,  and  as  a 
result  of  this  the  station  of  equilibrium  has  been  shifted. 

The  remarks  previously  made  upon  the  necessity  of  adequate 
dilution  hold  also  for  the  catalysor.  When  to  a  system  is  added 
an  excess  of  a  catalysor,  bizarre  and  irregular  results  may  be 
expected.  For  such  catalysors  as  act  through  ionization  this 
behavior  is  quite  intelligible.  It  is  here  again  possible  that  at  a 
high  concentration  of  the  catalysor,  it  will  in  part  participate  in 
the  solution  of  the  substrate.  Under  such  circumstances  the  solv- 
ent is  not  identical  with  that  in  a  system  at  high  dilution,  and 
since  the  station  of  equilibrium  may  be  shifted  very  materially 
by  alterations  in  the  solvent,  irregular  results  might  be  expected 
for  this  reason  alone.  Furthermore,  the  active  mass  of  the  sub- 
strate cannot  be  assumed  to  be  the  same  in  the  changed  state  of 
the  solvent.  There  is  in  any  event  no  necessity  for  the  use  of 
high  concentrations  of  the  catalysor,  since  one  of  the  most  strik- 
ing aspects  of  these  accelerations  is  the  minimal  amount  of  cata- 
lysor required  to  effect  a  notable  acceleration. 

When  to  a  system  are  added  several  catalysors,  what  happens  ? 
Their  accelerations  may  be  simply  superadded,  or  the  results 
may  be  greater  or  less  than  the  sum  of  their  individual  reac- 
tion velocities.  It  is  apparent  that  we  have  secondary  reac- 
tion to  deal  with,  through  which  alone  the  abnormalities  are  to 
be  explained.  In  some  instances  the  second  catalysor  is  formed 
as  a  product  during  the  course  of  the  reaction,  and  the  conse- 
quent increment  is  spoken  of  as  auto-catalysis.  A  good  illus- 
tration is  afforded  in  the  catalytic  acceleration  of  the  cleavage 
of  aethyl  acetate  into  aethyl  alcohol  and  acetic  acid.  Let  us  sup- 
pose that  the  entire  system  be  so  diluted  that  the  acid  acting  as 
a  catalysor  is  entirely  dissociated.  Now  as  the  acetic  acid  is  set 
free,  it  will  likewise  undergo  a  certain  dissociation,  and  the  con- 
centration of  hydrogen  ions  in  the  solution  will  be  thereby  grad- 
ually increased;  and  since  the  acceleration  is  proportional  to 


116  University  of  California  Publications.       [PATHOLOGY 

the  concentration  of  hydrogen  ions,  the  velocity  will  increase. 
This  increment  is  an  auto-catalysis.  Pure  nitric  acid  attacks 
copper  very  slowly,  but  the  reaction  gradually  becomes  more 
rapid  as  more  nitrous  acid  is  formed  in  the  reduction  of  the 
nitric  acid ;  here  nitrous  acid,  a  product  of  the  reaction  between 
copper  and  nitric  acid  acts  as  the  auto-catalysor. 

Under  the  term  inactivation  are  understood  currently  several 
different  relations.  The  slowing  of  the  reaction  under  the  influ- 
ence of  concentrated  products  is  a  manifestation  of  the  mass 
law,  and  ought  not  to  be  referred  to  as  an  inactivation  of  the 
ferment.  The  ferment  may  be  inactivated  by  union  with  some 
extraneous  body,  or  even  by  its  mere  presence ;  in  a  purely  chem- 
ical inactivation,  the  ferment  will  be  restored  to  power  when 
the  offending  condition  is  removed.  Lastly  the  ferment  may  be 
inactivated  by  hydrolysis  or  oxidation,  in  which  it  is  destroyed  as 
a  catalytic  agent. 

Under  certain  conditions  the  presence  of  a  substance  not 
itself  a  catalysor  will  enhance  the  acceleration  of  a  known  cata- 
lysor.  A  good  illustration  of  this  fact  is  contained  in  the  ob- 
servation of  Traube,  that  the  presence  of  a  trace  of  cupric  sul- 
phate, in  itself  inactive,  will  increase  enormously  the  acceleration 
of  the  reduction  of  hydrogen  peroxide  by  ferrous  sulphate.  The 
mechanism  of  such  zymoexcitation  is  entirely  unclear. 

As  there  are  accelerating  substances,  so  there  are  retarding 
substances.  We  must  here  distinguish  between  negative  cata- 
lysors  and  anti-catalysors.  A  negative  catalysor  would  be  a  sub- 
stance that  retards  the  natural  reaction,  what  might  be  called 
the  auto-reaction.  Anti-catalysors  are  substances  that  inhibit  in 
part  the  accelerating  action  of  a  positive  catalysor.  Without 
denying  that  genuine  negative  catalysors  exist,  it  is  certain  that 
the  bodies  described  as  such  have  been  anti-eatalysors.  Titoff 
practically  denies  that  true  negative  catalysors  exist,  and  Young 
has  drawn  the  same  conclusion  from  his  researches.  In  many 
inorganic  reactions  the  most  trifling  amounts  of  foreign  sub- 
stances may  act  as  pronounced  anti-catalysors,  and  there  can  be 
no  doubt  that  in  the  domain  of  ferments  such  influences  are  still 
more  numerous  and  potent. 


VOL.  i.]  Taylor. — On  Fermentation,  117 

The  station  of  equilibrium  in  a  reaction  of  reversible  charac- 
ter is  dependent  upon  certain  conditions.  In  the  ideal  sense  it 
applies  to  a  system  at  constant  temperature  in  a  high  and  con- 
stant dilution.  It  is  possible  experimentally  to  alter  the  station  of 
equilibrium  in  several  ways.  More  of  the  substrate  may  be  added, 
in  which  event  the  reaction  in  the  direction  of  the  right  will  again 
assume  the  leadership  until  a  new  balance  is  established.  Some 
of  the  substrate  may  be  removed,  in  which  event  the  reaction  in 
the  direction  of  the  left  will  be  accelerated  until  a  new  balance  is 
established.  The  products  may  be  removed  or  more  of  those 
bodies  added,  with  the  obvious  resultant  accelerations  in  the 
respective  reactions  until  new  equilibria  are  established.  Or 
only  one  of  the  products  may  be  added ;  thus  one  can  add  acetic 
acid  to  the  system  aethyl  acetate  4-  water  =  acetic  acid  +  aethyl 
alcohol  until  the  equilibrium  is  established  in  the  sense  that  there 
are  biit  two  bodies  present,  aethyl  acetate  and  acetic  acid.  The 
station  of  equilibrium  may  also  be  altered  by  dilution  or  concen- 
tration of  the  entire  system.  Alteration  in  temperature  may 
also  bring  with  it  an  alteration  in  the  station  of  equilibrium,  must 
indeed  if  the  reaction  be  endo-  or  exothermic.  To  these  we  must 
add  two  further  procedures  for  the  translocation  of  the  point  of 
equilibrium — reactions  between  ferment  and  components,  and 
reactions  between  solvent  and  product.  These  considerations  of 
alterations  in  the  station  of  the  equilibrium  are  of  great  impor- 
tance in  the  study  of  fermentations,  and  much  of  the  reigning 
confusion  has  been  due  to  neglect  of  them. 

Of  great  influence  is  the  nature  of  the  solvent,  the  velocity 
of  numerous  reactions  varying  widely  with  different  solvents. 
For  the  study  of  fermentations  of  biological  origin  this  is  of  little 
consequence,  since  water  is  the  invariable  solvent.  It  is  possible 
that  the  solvent  in  its  relations  to  the  concentration  of  the  react- 
ing bodies  may  be  influenced  by  the  presence  of  extraneous  sub- 
stances. We  know  that  in  very  concentrated  systems  the  sub- 
strate acts  in  part  as  solvent  for  the  catalysor;  and  in  high 
concentrations  of  the  catalysor  this  body  acts  as  a  solvent  for 
the  substrate.  In  either  event  the  point  of  equilibrium  would  be 
shifted. 


118  University  of  California  Publications.       [PATHOLOGY 

Purity  of  the  reacting  bodies  is  generally  of  great  importance 
for  the  regular  progression  of  catalytic  accelerations.  The  simple 
presence  of  foreign  bodies  is  not  itself  of  necessity  a  disturbing 
factor ;  extraneous  substances  may  be  divided  into  active  and  in- 
active. A  trace  of  an  active  substance  may  seriously  disturb  the 
reaction  in  an  otherwise  flawless  catalysis ;  a  good  illustration  is 
afforded  in  the  inhibition  that  may  be  exerted  upon  a  large  mass 
of  platinum  by  a  trace  of  arsenic,  in  the  contact  method  for  the 
manufacture  of  sulphuric  acid.  On  the  other  hand,  the  mere 
presence  of  one  or  a  number  of  inert  substances  may  have  little 
or  no  disturbing  effect.  We  shall  later  see  that  it  is  sometimes 
possible  to  secure  very  good  quantitative  results  in  fermentation 
experiments  conducted,  with  materials  known  to  be  very  com- 
plex. In  other  instances  good  results  are  not  secured,  the  differ- 
ence depending  not  upon  the  presence  of  foreign  substances,  but 
upon  the  presence  of  particular  foreign  substances  that  are  ac- 
tive in  their  chemical  relations  to  some  member  of  the  system 
under  investigation.  The  figures  obtained  by  Arrhenius  in  his 
studies  upon  the  quantitative  relations  between  biological  bodies 
and  anti-bodies  present  an  excellent  illustration  of  the  fact  that 
quantitative  measurements  may  sometimes  be  secured  under  con- 
ditions of  great  complexity  in  the  systems,  provided  only  that 
the  reactions  under  investigation  be  only  sufficiently  specific  and 
elective.  Nevertheless  it  were  always  best  to  work  under  pure 
conditions,  and  these  would  be,  in  the  case  of  the  subject  occu- 
pying our  attention,  a  pure  substrate,  dissolved  in  a  pure  solvent, 
and  the  reaction  accelerated  by  a  pure  catalysor.  The  best 
studies  in  the  literature  have  been  accomplished  under  such  con- 
ditions. In  the  various  chemical  investigations  upon  this  mat- 
ter it  has  been  acids,  alkalies  and  inorganic  salts  in  particular 
that  have  produced  disturbances  in  the  progress  of  catalytic  ac- 
celerations. A  priori  we  can  understand  that  since  we  conceive 
the  modus  operandi  of  catalytic  acceleration  to  lie  in  successive 
intermediary  reactions,  there  must  be  for  each  type  or  group  of 
reactions  certain  substances  whose  introduction  into  the  system 
would  lead  to  different  reactions  that  would  proceed  more  or 
less  at  the  expense  of  and  certainly  to  the  marked  disturbance 
of  quantitative  progression  in  the  original  reactions.  Just  as 


VOL.  l-l  Tai/lor. — On  Fermentation.  119 

there  are  certain  substances  and  conditions  that  prevent  a  good 
yield  in  the  synthesis  of  organic  substances,  and  these  conditions 
and  substances  vary  for  the  different  synthetic  procedures,  so 
there  are  conditions  and  substances  that  disturb  or  prevent  the 
regular  march  of  a  catalytic  acceleration,  and  these  conditions 
and  substances  will  vary  with  the  different  reactions  whose  accel- 
erations are  being  studied. 

Furthermore  irregularities  may  arise  in  the  reactions  when 
pure  substances  are  employed.  In  many  instances  the  reacting 
possibilities  in  a  system  are  not  limited  to  one  reaction  according 
to  one  formula,  but  may  permit  of  reactions  according  to  more 
than  one  formula.  Under  such  circumstances,  should  the  actual 
reaction  follow  other  than  the  reaction  according.to  one  formula, 
the  results  would  exhibit  deviations  more  or  less  removed  from 
the  velocity  demanded  by  the  simple  equation,  and  in  proportion 
to  the  extent  of  the  deviation  from  the  single  simple  reaction. 
In  other  instances  the  products  of  a  pure  reaction  themselves 
undergo  reactions  not  tending  to  a  reversion  into  the  original 
substrate.  Thus  the  products  may  react  with  each  other,  and 
then  a  totally  new  series  of  reactions  will  accompany  the  pri- 
mary reaction.  We  see  in  the  instance  of  the  acceleration  of  the 
reaction  S02  -|-  0  =  S03  through  the  simultaneous  presence  of 
the  reaction  f erro-salt  -)-  0  =  f erri-salt,  how  the  presence  of  an 
additional  reaction  may  alter  the  reaction  velocity;  and  such 
influence  may  be  conceivably  retardative  as  well  as  accelerating. 
Not  only  may  the  products  react  with  each  other ;  they  may  react 
with  the  substrate,  with  the  production  of  an  auto-catalysis  or 
auto-anti-catalysis;  or  they  may  in  other  ways  disturb  the  pro- 
gress of  the  reaction,  as  by  combining  with  the  substrate,  whereby 
the  concentration  of  the  active  mass  of  the  substrate  would  be 
altered.  Strictly  speaking,  reversion  of  the  reaction  and  auto- 
catalysis  are  to  be  classified  also  as  complicating  factors  that 
disturb  the  regular  progress  of  a  reaction  in  accordance  with 
the  general  law  of  mass  action,  and  systematic  authors  like  Ost- 
wald  do  so  classifv  these  conditions. 


120  University  of  California  Publications.       [PATHOLOGY 

PEESENT  APPLICATION  OF  KINETICS  OF  EEACTIONS  TO 
FEEMENTATIONS. 

When  now  we  apply  these  considerations  to  the  study  of  the 
natural  ferments  of  the  biological  world  we  experience  intel- 
lectual sensations  that  include  both  surprise  and  shock.  The 
literature  on  the  enzymes  is  enormous,  and  from  the  most  varied 
points  of  view  a  most  stupendous  amount  of  work  has  been  de- 
voted to  this  subject.  In  so  many  instances  however  has  the 
definition  of  the  work  been  so  uncontrolled  and  the  execution  so 
indefinite  that  the  results  have  in  no  wise  contributed  to  an 
actual  elucidation  of  the  relations  concerned.  Many  of  the  diffi- 
culties however  are  almost  inherent  in  the  nature  of  the  material. 
One  cannot  fail  further  to  note  in  the  biological  investigations 
of  fermentation  an  attitude  of  conscious  or  unconscious  antagon- 
ism to  the  physical  and  chemical  point  of  view  in  the  problem. 
It  must  be  realized  that  the  biological  and  medical  world  has  in 
large  part,  and  up  to  within  recent  date,  occupied  a  situation  of 
constitutional  inimicality  to  the  physico-chemical  interpretation 
of  biological  phenomena.  This  has  been  revealed  in  so  many 
investigations  of  fermentations.  The  physicist,  when  investigat- 
ing the  validity  of  a  law  in  a  series  of  phenomena  fixes  the  con- 
ditions of  experimentations  within  such  limits  as  have  been 
indicated  to  contain  the  naturally  favorable  and  controlled  rela- 
tions of  the  process  under  investigation.  In  much  of  the  study 
of  fermentations,  on  the  contrary,  the  method  of  procedure  has 
been  to  carry  out  the  experiments  not  in  the  narrow  zone  of 
controlled  conditions,  but  in  the  widest  zone  of  uncontrolled  con- 
ditions ;  and  then,  when  the  experimental  results  did  not  bear  out 
the  postulations  of  the  law,  to  denounce  the  law. 

Concentration  of  the  substrate.  This  is  in  fermentations 
often  difficult  to  adjust  and  determine.  Most  of  the  materials 
that  are  employed  as  the  substratum  of  fermentations  are  nat- 
ural substances,  and  therefore  never  pure.  Furthermore  it  is 
often  not  possible  to  determine  the  quantitative  degree  of  im- 
purity. Let  us  take  starch  for  illustration.  Starch  contains  a 
certain  amount  of  ash,  composed  of  various  inorganic  bodies.  It 
contains  also  organic  bodies,  traces  of  sugar,  fats,  protein  and 
what  not  more.  These  bodies  cannot  be  separated  from  starch, 


VOL.  l.]  Taylor. — On  Fermentation.  121 

and  the  only  way  of  determining  the  actual  amount  of  starch  is 
to  hydrolyse  completely  with  strong  acid  and  then  determine  the 
sugar  present,  and  even  this  method  would  neglect  the  preformed 
sugars.  When  therefore  one  prepares  a  1  per  cent,  solution  of 
starch,  one  does  not  know  with  just  how  much  under  a  1  per 
cent,  solution  one  is  actually  working.  But  more,  starches  vary 
in  their  resistance  to  hydrolytic  cleavages,  depending  upon  their 
origin,  mode  of  preparation,  age,  and  method  of  preservation. 
A  certain  starch  might  be  already  hydrolysed  to  as  much  as  10 
per  cent.,  according  to  the  method  of  preparation  and  preserva- 
tion. Let  us  further  consider  albumin.  All  that  has  been  said 
of  native  starch  holds  true  of  native  albumin  to  still  greater 
degree.  Not  only  are  the  foreign  substances  present  to  greater 
extent  and  in  greater  variety  than  in  starch,  but  the  tendency 
to  alteration  in  the  albumin  itself  is  more  pronounced.  Further- 
more an  albumin  is  usually  composed  of  several  proteins,  and 
unless  one  works  with  a  pure  protein  like  casein,  the  experiment 
actually  involves  the  digestion  of  several  proteins  of  possibly 
widely  varying  resistance  to  hydrolysis.  While  it  is  possible  in 
some  instances,  as  shown  by  Henri,  to  carry  along  in  the  same 
solution  two  catalyses  of  different  materials  and  have  each  main- 
tain its  own  proper  velocity  (the  inversion  of  sugar  and  the 
cleavage  of  methyl  acetate  by  acids),  it  would  not  be  possible 
to  do  so  under  circumstances  where  the  products  of  the  one  reac- 
tion make  the  measurement  of  the  other  reaction  uncertain  or 
futile.  One  could  certainly  not  hydrolyze  aethyl  acetate  and 
aethyl  succinate  in  the  same  solution  and  secure  anything  but 
irregular  results.  Yet  this  is  what  is  attempted  every  day  when 
egg  albumin  is  employed  as  the  substrate.  It  is  clear  therefore 
that  it  is  often  not  possible  to  fix  the  concentration  of  and  to 
exclude  a  plurality  in  the  substrate.  Now  since  the  concentra- 
tion of  the  active  mass  of  the  substrate  is  a  fixed  requirement,  it 
is  clear  that  at  the  very  outset  in  a  fermentation  we  are  con- 
fronted with  an  inability  to  define  properly  the  conditions  of  the 
experiment.  In  the  early  part  of  the  fermentation  this  indefi- 
nite minus  in  the  concentration  will  not  disturb  the  results  so 
seriously,  but  towards  the  close  of  the  reaction  the  influence  must 
be  marked.  This  state  of  affairs  is  of  course  not  general ;  we  can 


122  University  of  California  Publications.       [PATHOLOGY 

prepare  cane  sugar,  any  of  the  hexoses,  alcohols  and  the  synthetic 
fats  with  a  high  degree  of  purity,  and  it  has  been  along  these 
lines  that  much  of  the  best  work  has  been  done.  One  ought 
always  to  attempt  the  purification  of  the  other  materials,  but 
very  often  such  attempts  at  purification  only  lead  to  some  de- 
naturation  of  the  substance,  so  that  nothing  is  gained. 

Experience  with  fermentations  teaches  that  there  are  three 
zones  of  concentrations  with  particular  behaviors.     In  the  zone 
of  high  concentrations,  variations  lead  to  no  changes  in  velocity. 
In  a  system  with  an  excessive  concentration  of  substrate,  the  ve- 
locity depends  on  the  concentration  of  ferment  alone;  within 
certain  limits,  variations  in  the  substrate  have  no  result.     In  a 
system  with  an  excessive  concentration  of  ferment,  the  velocity 
depends  on  the  concentration  of  substrate  alone ;  within  certain 
limits  variations  in  the  concentration  of  ferment  have  no  result. 
Where  both  substrate  and  ferment  are  in  excessive  concentra- 
tion, irregular  and  bizarre  results  will  be  obtained.     In  the  zone 
of  medium  concentrations,  the  velocity  of  the  transformation  is 
a  function  of  the  mass  of  substrate,  and  the  intensity  of  the 
acceleration  is  a  function  of  the  concentration  of  ferment,  but 
there  is  an  interdependence.     The  intensity  of  the  fermentative 
acceleration  accomplished  by  a  constant  concentration  of  ferment 
will  vary  to  some  extent  with  the  concentration  of  the  substrate. 
For  different  concentrations  of  substrate  the  ferment  seems  to 
set  a  different  pace,  a  condition  that  suggests  a  sort  of  a  stoichio- 
metric  relation.    To  this  variation  in  intensity  of  ferment  action 
Henri1  and  Visser  attach  a  high  importance,  to  which  reference 
shall  be  made  later.     Most  of  the  experiments  with  ferments 
have  been  conducted  within  this  zone  of  concentrations.     In  the 
last  zone  of  concentration,  that  of  high  dilutions,  the  velocity  of 
the  transformation  is  the  function  of  the  substrate  concentration, 
and  the  intensity  of  the  acceleration  is  the  function  of  the  mass 
of  ferment;  and  these  are  independent,  just  as  they  should  be 
in  a  pure  catalysis.     When  operating  within  this  zone,  dilution 
of  the  entire  system  has  no  effect  upon  the  proportionalities  of 
the  reaction,  while  the  contrary  is  observed  on  dilution  of  the 
systems  in  the  other  zones  of  concentration.     I  believe  that  if 
the  proper  conditions  can  be  attained,  this  zone  will  be  found  in 


VOL.  1.]  Taylor. — On  Fermentation.  123 

many  fermentations.  For  the  study  of  fermentation  from  the 
kinetic  point  of  view,  this  zone  of  concentration  is  obviously  the 
one  to  be  sought. 

It  must  be  insisted  upon  that  the  failure  of  the  law  of  mass 
action  to  hold  within  wide  limits  of  concentrations  is  not  peculiar 
to  the  phenomena  of  fermentations.  For  many  of  our  best  known 
physico-chemical  laws  the  conformity  of  fact  to  theory  is  con- 
fined to  narrow  limits  of  conditions.  Van't  Hoff  3  in  the  dis- 
cussion of  the  general  equation  for  simple  reactions,  — 


dt 

kCi  Cu  .  .  ,  remarks  that  the  equation  is  valid  only  for  quite 
high  dilutions,  practically  tenth  normal.  The  law  of  electro- 
lytic dissociation  applies  only  to  conditions  of  infinite  dilution, 
and  the  variations  that  are  induced  by  the  presence  in  the  system 
of  undissociated  molecules  are  well  illustrated  in  the  recent  in- 
vestigation of  Jahn.  Similar  relations  were  naturally  to  have 
been  expected  in  fermentations,  and  more,  for  the  multiplicity 
of  the  conditions  should  lead  us  to  expect  the  relations  here  to  be 
still  much  more  complex. 

A  further  consideration  must  be  emphasized.  The  total  con- 
centration of  the  substrate  must  be  so  low  that  the  entire  mass 
of  the  solution  shall  be  active  in  the  sense  of  the  mass  law.  But 
there  is  in  catalytic  accelerations  possibly  a  further  extension 
of  the  concept  "active"  necessary.  For  the  catalytic  accelera- 
tion, only  that  fraction  of  the  substrate  may  be  considered  active 
which  in  combination  with  the  catalysor  is  engaged  in  the  series 
of  intermediary  reactions  that  we  believe  constitute  the  modus 
operandi  of  these  accelerations.  This  depends  obviously  on  a 
certain  relationship  between  the  concentrations  of  substrate  and 
ferment.  In  the  zone  of  high  dilution  of  substrate  and  moderate 
dilution  of  ferment  alone  may  we  expect  to  find  the  condition 
that  all  of  the  substrate  shall  be  in  relation  to  ferment,  and  thus 
active  in  the  full  sense ;  here  alone  the  law  can  be  fulfilled.  To 
what  extent  this  consideration  enters  into  the  factor  of  ' '  ferment 
intensity ' '  is  not  known.  That  the  fact  holds  for  pure  catalyses 
may  be  seen  in  the  acid  hydrolysis  of  starch  and  protein,  where 
quantitative  results  may  be  secured  that  are  very  similar  to  those 
seen  in  fermentations.  In  the  acid  inversion  of  disaccharides 
the  disturbances  are  not  noted,  but  in  the  acid  hydrolysis  of  the 


124  University  of  California  Publications.       [PATHOLOGY 

complex  starch  the  phenomena  are  as  observable  as  in  fermen- 
tations. 

Armstrong,  in  his  discussion  of  his  results  of  the  study  of  the 
inversion  of  disaccharides,  has  lucidly  stated  the  conditions  that 
are  to  be  expected  with  different  relations  of  concentrations.  His 
statements  may  be  quoted  in  some  detail. 

' '  The  proportion  of  the  total  substrate  present  combined  with 
the  enzyme  and  undergoing  change  at  any  one  moment  may  be 
regarded  as  the  active  mass  of  the  substrate.  *  *  *  On  the 
hypothesis  that  the  enzyme  combines  with  the  substrate,  the  ac- 
tive mass  of  the  latter  will  be  that  portion  of  the  whole  S  which 
is  in  combination  with  an  amount  of  enzyme  e;  it  will  be  con- 
venient to  speak  of  the  combination  s  -\-  e  as  the  active  system. ' ' 
He  then  proceeds  to  discuss  the  possible  relations.  "Case  I,  in 
which,  whatever  the  amount  of  substrate  present,  the  quantity 
of  enzyme  is  relatively  small.  Case  II,  in  which  there  is  a  dif- 
ference from  Case  I,  inasmuch  as  the  quantity  of  enzyme  is 
relatively  considerable.  Case  III,  in  which  the  amount  of  en- 
zyme diminishes  as  the  action  proceeds.  Case  IV,  in  which  the 
amount  of  substrate  is  varied.  Case  I.  As  action  proceeds,  since 
the  magnitude  of  the  active  system  depends  on  the  amount  of 
the  enzyme  present,  it  is  obvious  that,  in  the  initial  stages,  if  the 
total  amount  of  substrate  S  be  large  compared  with  s,  the  enzyme 
will  be  in  presence  of  enough  substrate  molecules  to  establish 
the  maximum  possible  number  of  effective  combinations ;  or  in 
other  words,  the  magnitude  of  the  active  system  will  remain  con- 
stant and  the  change  will  be  expressible  as  a  linear  function  of 
the  time.  As  the  action  proceeds  further,  the  amount  of  S  of 
substrate  present  decreases  until  it  is  no  longer  negligible  com- 
pared with  that  of  the  active  part  s,  and  hence  the  enzyme  will 
no  longer  effect  the  maximum  possible  number  of  combinations ; 
the  proportion  s  undergoing  change  will  then  be  a  function  of 
the  total  mass,  and  the  formation  of  active  systems  will  be  gov- 
erned by  the  law  of  mass  action.  The  rate  of  change  will  be  a 
logarithmic  function  of  the  time.  Case  II.  If,  on  the  other 
hand,  the  quantity  of  enzyme  used  be  relatively  large,  the  active 
mass  will  be  a  function  of  the  total  mass  from  the  very  begin- 
ning of  the  experiment,  so  that  the  linear  part  of  the  change  will 


VOL.  1.]  Taylor. — On  Fermentation.  125 

escape  notice.  *  *  *  Stated  shortly,  the  law  of  mass  action 
is  applicable  only  to  the  period  during  which  a  constant  rela- 
tively large  proportion  of  enzyme  is  present  together  with  a  con- 
tinually decreasing  amount  of  substrate,  but  uninfluenced  by  the 
products  of  change."  Case  III  does  not  concern  us 

here.  "Case  IV.  When  the  amount  of  enzyme  and  water  is 
kept  constant,  while  that  of  the  substrate  is  increased,  it  may  be 
supposed  that  the  magnitude  of  the  active  system  will  increase 
until  s  -(-  e  reaches  a  maximum,  a  definite  equilibrium  being 
established  between  enzyme,  substrate  and  water,  the  whole  of 
the  enzyme  perhaps  being  combined  with  the  substrate.  *  *  * 
If  s  -\-  e  remains  unaltered,  whatever  the  proportion  of  substrate 
present  beyond  a  certain  minimum,  a  constant  amount  of  sub- 
strate will  undergo  change  in  a  given  time. ' ' 

Measurement  of  the  reaction.  Frequently  we  are  not  in  a 
position  to  measure  in  an  accurate  analytic  manner  the  products 
of  the  reaction.  It  is  rare  that  we  have  the  opportunity  to 
measure  the  products  with  a  poloriscope,  by  a  direct  accurate 
titration  or  by  a  determination  of  conductivity.  More  often  we 
need  to  undertake  a  formal  isolation  of  the  desired  product  and 
then  identify  it  and  measure  it  by  means  of  some  chemical  be- 
havior. In  some  instances  such  a  procedure  may  yield  accurate 
results,  but  it  may  not  be  feasible  to  do  this  in  the  number  of 
determinations  necessary  in  a  measurement  of  reaction  velocity. 
Sometimes  the  product  may  not  be  accurately  estimated  in  any 
way,  as  in  the  case  of  the  digestion  of  protein. 

A  more  serious  complication  arises  with  a  great  many  fer- 
mentations in  which  the  reaction  occupies  not  one  but  many 
stages.  These  mediated  catalyses  exceed  the  direct  catalyses  in 
the  organic  world.  We  have  substrate  -(-  water  —  produc^  -(- 
produc^  ;  produc^  -f-  water  =  p2  -(-  P2 ;  etc.,  etc.,  and  finally  pn 
-\-  water  =  endproduct  -{-  endproduct.  These  really  represent  a 
series  of  successive  catalyses.  The  reactions  however  do  not 
progress  in  complete  stages;  that  is,  all  of  the  substrate  is  not 
first  converted  into  produc^ ;  and  all  of  product!  then  converted 
into  product,  and  so  on  until  the  endproducts  are  reached.  On 
the  contrary,  several  of  the  products  may  be  found  in  Jjie  mix- 
ture in  a  particular  moment.  Each  of  these  stages  represents 


126  University  of  California  Publications.       [PATHOLOGY 

work.  Now  in  a  fermentation  experiment  under  such  conditions, 
what  shall  one  measure?  Obviously  if  one  wished  to  measure 
the  reduction  in  the  substrate  one  must  measure  product^  which 
cannot  be  done  because  the  stage  is  not  completed  en  bloc.  If 
one  measures  the  endproduct,  what  one  really  measures,  as  Henri 
has  pointed  out,  is  the  appearance  of  the  endproduct  and  not 
the  conversion  of  the  substrate.  And  yet  the  measurement  of 

the  endproduct  may  be  only  possible  measurement.    Under  these 

1  A 

circumstances  Henri  rearranges  the  equation  C  =  -  log.  ^ — 

1 —  (A          X) 

so  that  under  A  we  understand  the  quantity  of  endproduct  when 
the  reaction  is  completed  and  under  x  the  quantity  of  endpro- 
duct formed  in  the  time  t.  Under  these  circumstances  it  be- 
comes possible  to  work  with  fermentations  involving  reactions 
in  many  stages,  though  in  some  instances  the  results  are  very 
irregular.  It  is  apparent  that  while  a  positive  result  would  indi- 
cate that  the  law  holds  good  for  the  particular  fermentation,  a 
negative  result  would  not  indicate  the  converse;  it  would  indi- 
cate simply  nothing,  beyond  that  the  conditions  of  experimenta- 
tion were  too  uncontrolled  to  yield  results  susceptible  of  inter- 
pretation. The  majority  of  the  fermentations  of  great  biological 
importance  belong  to  this  class  of  mediated  catalyses;  no  highly 
organized  body  like  cellulose,  starch,  protein,  may  be  hydrolyzed 
into  simple  chrystalline  endproducts  in  a  single  main  reaction. 

It  must  be  realized  that  this  constitutes  not  an  analytical  but  a 

(I  i' 
fundamental  difficulty.   The  equation  — -— -=  C  (A  —  x)  presup- 

Clt 

poses  that  the  substance  undergoing  transformation  exists  in 
each  moment  in  the  system  either  in  the  form  of  unaltered  sub-, 
strate  A  or  of  product  x.  The  intermediary  reactions  from  the 
state  A  to  x  are  not  held  to  occupy  measurable  time.  If  now  the 
substrate  in  the  moment  of  analysis  exists  in  part  in  the  state  of 
e,  f,  or  more  states,  and  not  either  as  A  or  x,  the  equation  cannot 
apply.  This  is  precisely  what  occurs  in  the  digestion  of  complex 
substances  like  protein  and  starch,  and  under  these  circumstances 
one  cannot  be  surprised  that  the  fermentations  of  these  sub- 
stances do  not  follow  closely  the  law  of  mass  action  as  expressed 
in  the  equation. 

Reversion  of  the  Reaction. — The  equation  for  the  simple  mo- 
nomolecular  reaction  does  not  contemplate  a  reversion  of  the 


VoL-  !•]  Taylor. — On  Fermentation.  .       127 

reaction.  For  fermentations,  however,  we  postulate  theoreti- 
cally such  a  reversion.  If  this  reversion  occur  with  anything 
like  measurable  rapidity,  the  constants  obtained  with  the  use  of 
the  equation  cannot  be  identical  and  an  entirely  different  mathe- 
matical formulation  will  be  necessary.  That  a  certain  degree  of 
reversion  occurs  in  actual  fermentations  has  been  practically  as- 
sumed on  the  basis  of  three  facts :  the  substrate  is  never  com- 
pletely fermented;  removal  of  the  products  increases  the  com- 
pleteness of  the  fermentation,  and  reinaugurates  it  after  it  has 
ceased;  and  the  removal  of  the  products  increases  the  rapidity 
of  the  fermentation.  It  must,  however,  not  be  overlooked  that 
two  of  these  results  could  be  due  to  chemical  influence  of  the 
products  upon  the  ferment.  As  a  matter  of  fact,  the  results  of 
the  many  known  experiments  in  reversions  by  ferment  action 
(in  which  the  ferment  has  been  mixed  with  the  products  of  the 
reaction)  has  been  to  indicate  that  reversions  occur  with  great 
slowness  as  compared  to  the  reaction  in  the  other  direction.  If 
one  attempts  to  incorporate  into  the  equation  of  a  monomolecular 
reaction  as  a  conditioning  factor  that  rapidity  of  reversion  ex- 
perimentally observed  in  direct  tests,  the  deviation  will  be  almost 
nil.  In  addition  to  this,  the  station  of  equilibrium  is  for  most 
substrates  so  near  to  a  completed  reaction  that  reversion  could 
scarcely  be  held  to  modify  to  any  appreciable  extent  the  mathe- 
matical expression  of  a  ferment  reaction.  Nevertheless,  the  act- 
ual solution  of  the  extent  of  this  variable  lies  not  in  mathemat- 
ical considerations,  but  in  the  direct  experiment.  It  may  be  one 
of  the  reasons  why  the  experimental  velocities  in  fermentations 
do  not  always  agree  with  the  mathematical  predications.  To  the 
factor  of  reversion  special  attention  has  been  called  by  Visser, 
and  we  shall  consider  his  studies  in  detail  later. 

Concentration  of  Ferment.  It  is  as  difficult  to  obtain  and 
maintain  a  constant  concentration  of  the  ferment  as  of  the  sub- 
strate. The  activities  of  ferments  as  well  as  their  stabilities 
seem  to  depend  to  a  marked  degree  upon  the  methods  of  prepa- 
ration and  conservation.  All  ferments  contain  more  or  less 
extraneous  matter ;  probably  in  most  instances  the  percentage 
of  the  actual  ferment  is  but  a  small  fraction  of  the  weight  of 
the  preparation  employed.  Under  such  circumstances  it  is  not 


128  University  of  California  Publications.       [PATHOLOGY 

possible  to  define  the  exact  initial  concentration  of  ferment,  and 
this  results  in  an  indeterminate  error  in  the  course  of  series 
of  experiments. 

The  lack  of  regularity  between  the  chemical  composition  and 
enzymic  activity  of  a  ferment  is  a  condition  not  peculiar  to  fer- 
ments, but  is  an  unfortunate  quality  of  all  colloids,  for  the  desig- 
nation of  which  van  Bemmelen  has  amplified  the  use  of  the  term 
hysterisis.  The  age,  origin,  method  of  preparation  and  in  short 
every  incident  in  the  history  of  a  colloid  tend  to  influence  its 
chemical  qualities.  Further  than  this,  once  prepared  and  con- 
served under  constant  condition,  colloids  tend  slowly  to  altera- 
tions that  may  in  general  be  described  as  changes  towards  crys- 
talloid qualities.  This  denaturation  could  be  properly  con- 
ceived to  lead  to  a  reduction  in  the  dynamically  active  mass  of 
the  colloid. 

Of  far  greater  importance  than  the  difficulty  in  determining 
the  initial  concentration  is  the  impossibility  of  maintaining  the 
concentration.  Ferments  as  a  class  have  been  held  to  differ  from 
the  inorganic  catalysors  in  that  the  latter  emerge  from  the  com- 
pleted reaction  unchanged.  This  is  not  strictly  true,  for  the 
colloid  metals  are  subject  to  similar  reduction  in  their  catalytic 
properties.  In  some  instances  the  inactivation  is  accompanied  by 
precipitation  in  a  granular  amorphous  form,  but  in  other  in- 
stances the  appearances  of  the  colloid  have  undergone  no  change. 
The  inactivation  proceeds  more  rapidly  at  high  temperatures, 
and  seems  to  affect  old  suspensions  more  than  fresh  preparations. 

All  ferments  are  more  or  less  altered  during  the  course  of  a 
fermentation.  Upon  the  supposition  that  we  are  dealing  with 
pure  conditions,  there  are  apparently  three  conceivable  sources 
for  these  alterations,  namely:  reactions  with  the  substrate,  the 
products,  or  with  the  solvent.  We  have  almost  no  knowledge 
of  reactions  between  the  substrate  and  ferment  that  would  re- 
sult in  inactivation  of  the  ferment.  Macintosh  has  furnished  a 
good  illustration  of  this  relation  in  the  acceleration  of  the  re- 
duction of  hydrogen  peroxide  by  colloidal  silver.  The  substrate 
combines  with  a  portion  of  the  silver  to  form  a  compound  that 
is  catalytically  inactive.  We  have  little  knowledge  of  the  inac- 
tivation of  ferments  by  reactions  with  the  products.  I  have- 


VOL.  1.]  Taylor. — On  Fermentation.  129 

determined  that  trypsin  is  more  rapidly  destroyed  in  a  solution 
of  amido-acids  (products  of  tryptic  digestion)  than  in  simple 
watery  solution.  Vegetable  lipase  is  somewhat  sensitive  to  acids, 
and  is  destroyed  more  rapidly  in  even  moderate  concentrations 
of  acetic  acid  than  in  distilled  water.  In  all  probability  how- 
ever the  chief  reaction  resulting  in  the  destruction  of  the  fer- 
ment is  hydrolysis  upon  the  part  of  the  solvent.  Just  as  the 
albumin  or  ester  constituting  the  substrate  undergoes  on  suspen- 
sion in  water  a  slow  hydrolytic  cleavage  of  which  the  digestion 
is  but  the  acceleration,  so  ferments  on  suspension  in  water  un- 
dergo hydrolysis ;  and  as  the  products  are  not  active  in  the  cata- 
lytic sense,  the  active  concentration  of  the  ferment  is  thus  steadily 
reduced  during  the  progress  of  the  experiment.  The  two  hydro- 
lyses,  of  the  substrate  and  of  the  ferment,  proceed  side  by  side, 
probably  entirely  independently.  The  hydrolysis  of  the  ferment 
is  of  course  accelerated  by  increase  in  temperature,  and  seems 
to  follow  the  usual  rule  for  such  increase.  I  have  found  that 
the  destruction  of  vegetable  lipase  proceeds  closely  according  to 
this  rule,  while  the  destruction  of  trypsin  proceeds  rather  more 
rapidly.  The  possible  extent  of  such  an  inactivation  will  be 
understood  when  one  realizes  that  in  some  instances  a  solution 
of  trypsin  may  be  inactivated  one-fifth  in  an  hour;  and  the  de- 
gree of  disturbance  that  must  follow  becomes  apparent  when  we 
consider  that  some  half  dozen  hours  might  be  required  for  a 
digestion  experiment  with  such  a  •  solution.  In  their  resistance 
to  hydrolysis  ferments  however  vary  widely.  The  inactivatiou 
of  the  ferment  proceeds  usually  more  rapidly  in  simple  solution 
in  water  than  during  the  course  of  a  fermentation  experiment; 
this  has  been  shown  true  for  amylase,  invertase,  trypsin,  vege- 
table lipase,  and  for  zymase.  The  natural  interpretation  of  this 
phenomenon  is  that  during  the  course  of  the  fermentation  the 
ferment  is  passing  through  intermediary  reactions  with  the  sub- 
strate, and  that  when  thus  combined  its  hydrolysis  is  suspended. 
The  free  ferment  adds  water  to  undergo  cleavage;  in  the  com- 
plex substrate-ferment  this  reaction  does  not  occur. 

Tammann,  who  first  studied  this  matter,  attempted  to  allow 
for  this  inactivation  by  the  insertion  of  a  corresponding  factor 
in  the  formula  for  reaction  velocity.  Upon  the  assumption  that 


130  University  of  California  Publications.       [PATHOLOGY 

the  reaction  in  each  moment  is  proportional  to  the  momentary 
concentration  of  the  substrate  and  of  the  unaltered  ferment,  the 
velocity  would  be  expressed  in  the  following  equation : -^r  ~ 

QfT 

C  (A — x)  (F  —  y}.  F  is  the  original  mass  of  ferment,  y  the 
ferment  inactivated  in  the  time  t,  the  other  signs  are  as  before. 
Tammann  measured  the  destruction  of  emulsine  on  heating  dry 
and  in  solution,  and  found  the  reaction  in  water  to  be  of  the 
first  order.  The  data  for  the  destruction  of  the  ferment  he  then 
inserted  into  the  equation  above  given,  following  which  the  con- 
stant for  the  main  reaction  could  be  calculated.  Tammann 
made  the  statement  that  the  constant  for  the  destruction  of  the 
ferment  is  independent  of  the  ferment  concentration,  and  the 
same  for  all  preparations.  This  statement  is  at  variance  with 
the  truth ;  the  destruction  of  a  ferment  by  heat  varies  much  with 
the  history  of  the  preparation. 

This  formula,  however,  fails,  because  the  velocity  of  hydroly- 
sis of  the  ferment  is  not  the  same  during  the  course  of  a  fermen- 
tation as  by  itself,  but  is  less.  On  account  of  the  fact  that  the 
ferment  when  combined  with  the  substrate  is  resistant  to  hydro- 
lysis, one  must  for  the  purposes  of  the  calculation  of  the  hydroly- 
sis of  the  ferment,  distinguish  between  the  total  concentration  of 
the  ferment  and  the  active  mass,  the  latter  being  represented  by 
that  portion  of  the  ferment  not  in  combination  with  the  sub- 
strate. In  general  terms  therefore,  the  active  mass  of  the  cata- 
lysor  for  the  reaction  of  its  own  hydrolysis  would  be  inversely 
to  the  active  mass  of  the  substrate  of  the  main  reaction — the 
hydrolysible  catalysor  would  increase  proportionally  as  the  sub- 
strate diminishes,  and  the  velocity  of  the  destruction  of  the  fer- 
ment would  be  inversely  proportional  to  the  mass  of  the  sub- 
strate. As  the  fermentation  progresses,  the  velocity  of  the  hydro- 
lysis of  the  ferment  will  approach  the  velocity  noted  in  the  direct 
test,  because  the  concentration  of  the  substrate  is  steadily  dimin- 
ishing, and  upon  this  substrate  the  repression  of  the  hydrolysis 
depends.  While  the  cleavage  of  the  substrate  is  following  a 
logarithmic  curve  downwards,  the  hydrolysis  of  the  ferment  is 
following  a  logarithmic  curve  upwards.  And  since  upon  the 
basis  of  the  available  experimental  data  this  deviation  cannot  be 


VoL-  L]  Taylor. — On  Fermentation.  131 

experimentally  determined,  the  results  of  the  calculations  do  not 
in  practice  conform  to  the  findings. 

Inactivation  of  the  ferment  may  be  produced  also  by  reac- 
tions with  extraneous  substances.  Theoretically  it  ought  always 
to  be  possible  to  distinguish  between  inactivation  and  destruc- 
tion of  a  ferment;  practically  this  may  not  be  always  possible. 
The  phenomenon  is  very  frequent  in  actual  experimental  work, 
and  may  also  be  provoked  in  inorganic  catalyses.  The  accelera- 
ting action  of  iron  salts  may  be  abolished  by  the  presence  of 
acetic  or  oxalic  acidj  the  accelerating  action  of  colloidal  plati- 
num is  depressed  by  hydrocyanic  acid  or  a  thiosulphate  as  well 
as  by  alkalies,  etc.  These  influences  are  not  all  of  one  nature; 
in  some  the  ferment  or  catalysor  is  destroyed ;  in  others  the  action 
is  inhibited  though  the  substance  is  not  altered,  for  when  the 
depressing  body  is  removed  the  original  activity  of  acceleration 
returns.  Colloidal  platinum  again  affords  a  striking  illustration : 
the  inhibition  of  its  catalytic  acceleration  by  hydrocyanic  acid, 
carbon  monoxide  or  phosphorus  will  pass  away  with  the  re- 
moval of  those  substances,  while  the  inhibition  following  the 
addition  of  sulphuretted  arsenic,  iodine  or  mercuric  chloride 
remains  after  the  removal  of  those  bodies.  For  the  natural  fer- 
ments the  negatively  catalytic  influences  are  exceedingly  numer- 
ous, and  in  nearly  all  instances  they  effect  a  permanent  inactiva- 
tion of  the  ferments.  Many  of  these  substances  act  undoubtedly 
by  accelerating  the  hydrolytic  cleavage  of  the  ferment,  that  is, 
they  are  positive  catalysors  to  the  hydrolysis  of  the  ferment. 
Such  is  almost  certainly  the  nature  of  the  influence  of  acids  and 
alkalies. 

Stimulation  of  the  ferment  by  the  presence  of  substances  not 
in  themselves  accelerators  is  very  frequently  observed  in  con- 
nection with  fermentations.  Thus  a  trace  of  acid  aids  the  action 
of  invertase,  vegetable  lipase,  and  of  the  ferments  of  the  pepsin 
group;  a  trace  of  alkali  aids  many  reductions  and  also  the  fer- 
mentations of  the  trypsin  group.  Many  salts  have  similar  ac- 
tions, as  have  innumerable  other  substances.  These  zymo-excitors 
seem  to  have  two  things  in  common :  an  optimum  concentration 
and  an  optimum  temperature.  A  good  illustration  of  these  facts 
(and  one  that  has  the  further  value  that  it  also  illustrates  the 


132  University  of  California  Publications.       [PATHOLOGY 

identity  of  the  conditions  in  the  organic  and  inorganic  worlds) 
is  to  be  found  in  the  zymo-excitation  of  alkali  upon  the  reduction 
of  hydrogen  peroxide  by  colloidal  platinum  and  oxydase  of  ani- 
mal origin ;  for  both  of  these  reactions  one  may  obtain  a  curve 
of  the  influence  of  increasing  alkali-content  with  a  well  defined 
maximum,  and  for  different  alkali-content  also  a  curve  of  tem- 
perature influence  with  a  well  defined  maximum.  Some  in- 
stances of  zymo-depression  and  zymo-stimulation  will  be  consid- 
ered in  detail  in  connection  with  the  discussion  of  particular 
fermentations. 

Station  of  equilibrium.  We  have  to  deal  here  with  a  most 
interesting  phase  of  the  question  of  fermentations.  Pure  posi- 
tive catalysors  do  not  bring  about  any  translocation  of  the  sta- 
tion of  equilibrium.  When  such  a  thing  occurs  in  an  inorganic 
catalysis  it  is  because  some  one  of  the  possible  factors  mentioned 
has  intervened.  But  in  the  domain  of  fermentations  we  encoun- 
ter a  new  state  of  affairs,  namely  that  reactions  in  themselves 
practically  complete  and  unlimited,  and  which  remain  practi- 
cally complete  reactions  when  accelerated  by  inorganic  cata- 
lysors like  acids,  seem  to  become  limited  reactions  when  accel- 
erated by  ferments.  Many  ferments  seem  practically  unable  to 
carry  through  their  accelerations  to  the  point  of  complete  con- 
version of  the  substrate  into  the  products  that  is  observed  when 
inorganic  catalysors  are  employed.  This  is  not  true  of  all  fer- 
ments, but  of  a  great  many;  and  the  phenomenon  is  especially 
observed  in  the  hydrolysis  of  starch,  glucosides  and  protein.  The 
condition  was  first  described  by  Tammann  when  working  with 
amygdaline,  although  the  direct  observation  of  the  limited  con- 
version of  starch  had  been  made  by  Pay  en. 

The  facts  are  as  follows :  A  certain  reaction  when  accelerated 
with  hydrogen  ions  is  practically  a  complete  reaction.  Let  us 
say  the  condition  of  the  system  may  be  represented  by  the  rela- 
tions substrate  1 :  products  99.  At  this  point  the  reactions  in 
each  direction  are  equal;  the  tendency  to  combination  upon  the 
part  of  the  products  is  very  slight  since  with  all  the  mass  of 
products  the  combination  is  only  able  to  balance  the  cleavage 
of  the  substrate  when  its  mass  has  fallen  to  1  per  cent.  When 
this  same  reaction  is  accelerated  by  a  ferment  the  cleavage  may 


VOL.  i.]  Taylor.— On  Fermentation.  133 

not  be  nearly  so  complete,  and  at  the  close  the  relations  may  be 
expressed  something  like  this :  substrate  15 :  products  85.  Does 
this  apparent  shifting  in  the  point  of  equilibrium  correspond  to 
a  real  translocation  of  the  station  of  equilibrium,  to  a  change  in 
the  constant  of  equilibrium?  There  are  many  facts  that  tend 
to  indicate  that  such  may  be  really  the  case.  The  addition  of 
more  substrate  to  the  mixture  after  the  reaction  has  ceased  to 
progress  will  serve  to  reinaugurate  the  reaction.  When  the  pro- 
ducts of  the  reaction  are  added  early  in  the  course  of  the  experi- 
ment, the  reaction  will  cease  sooner  than  otherwise,  and  cease 
sooner  proportionately  to  the  quantity  of  products  added.  On 
the  other  hand,  when  the  products  are  removed  from  the  system, 
the  reaction  will  be  reinaugurated.  Concentration  or  dilution 
of  the  volume  will  also  disturb  the  apparent  equilibrium,  and 
an  increase  of  temperature  will  cause  the  reaction  to  recommence 
in  a  system  that  had  become  quiescent.  Finally  the  addition  of 
further  ferment  may  reinaugurate  the  reaction,  although  it  can 
be  easily  shown  that  an  abundance  of  active  ferment  was  still 
present  in  the  mixture.  The  only  possible  direct  interpretation 
of  the  last  fact  is  that  the  ferment  is  reacting  with  the  compon- 
ents of  the  system,  probably  with  the  substrate,  and  possibly  in 
a  relation  of  definite  proportions.  By  repeated  additions  of  fer- 
ment it  may  be  possible  in  some  instances  to  complete  the  reac- 
tion, provided  only  that  the  initial  concentration  of  the  system 
was  sufficiently  diluted.  These  various  facts  are  identical  with 
those  that  hold  for  a  translocation  of  the  station  of  equilibrium 
in  a  true  reaction  of  measurable  reversibility,  except  that  in 
these  cases  the  further  addition  of  catalysor  will  not  shift  the 
point  of  equilibrium.  The  least  difficult  interpretation  is  that 
the  ferment  has  entered  into  reactions  with  the  components  of 
the  original  system,  that  the  station  of  equilibrium  has  been 
shifted  thereby  and  that  the  reaction  to  the  left,  the  reversion, 
is  so  greatly  augmented  as  to  maintain  the  new  equilibrium.  As 
Bredig-  has  succinctly  expressed  it,  the  equilibrium  is  not  deter- 
mined by  the  substrate  and  the  products,  but  by  the  substrate, 
the  products  and  the  ferment.  The  exact  nature  of  the  phenom- 
enon is  not  at  all  clear.  As  a  matter  of  fact,  the  observation  has 
not  been  confined  to  ferments,  for  as  Musculus,  Wohl  and  others 


134  University  of  California  Publications.       [PATHOLOGY 

have  shown,  under  certain  conditions  the  acid  hydrolysis  of 
starch  is  not  complete,  and  in  fact  the  reversion  of  this  acid 
hydrolysis  has  been  made  highly  probable. 

When  to  a  reaction  naturally  limited  and  measurably  revers- 
ible one  adds  a  ferment,  how  is  the  equilibrium  affected  ?  There 
has  been  very  little  work  done  upon  this  class  of  reactions.  Ob- 
viously a  ferment  could  by  reacting  with  the  components  of  the 
system*  alter  the  constant  of  equilibrium  as  readily  as  in  the  case 
of  a  reaction  that  is  naturally  quite  complete.  I  have  studied 
the  question  by  experimentation  upon  various  esters  with  a  vege- 
table lipase,  and  I  have  never  been  able  to  find  that  the  constant 
of  equilibrium  has  been  shifted.  Apparently  this  lipase  at  least 
acts  as  a  pure  catalysor.  The  details  of  these  experiments  will 
be  furnished  later. 

CATALYSIS  IN  HETEEOGENEOUS  SYSTEMS. 

What  has  been  said  heretofore  applies  specifically  only  to 
homogeneous  systems.  Now  in  many  reactions  of  fermentation, 
as  well  as  in  many  organic  catalyses,  we  have  to  deal  with  heter- 
ogeneous systems,  and  very  important  physical  deviations  are 
here  presented.  We  have  furthermore  to  deal  with  different 
combinations  of  conditions.  The  substrate  may  be  solid,  the 
solvent  fluid  and  the  catalysor  fluid.  Or  the  substrate  may  be 
in  homogeneous  solution,  while  the  catalysor  may  be  solid.  There 
are  even  conditions  in  which  the  products  may  be  heterogeneous, 
as  in  the  enzymic  reaction  in  which  amorphous  sulphur  is  pro- 
duced from  hyposulphite.  And  lastly  we  have  the  condition, 
common  in  the  world  of  living  matter,  of  a  suspended  colloidal 
catalysor  accelerating  the  reaction  of  a  colloidal  substrate  sus- 
pended in  water. 

Let  us  first  consider  the  relations  when  the  substrate  is  col- 
loidal or  solid,  the  catalysor  fluid.  When  such  a  body  is  sus- 
pended in  water,  the  same  relations  will  hold  that  were  described 
by  Noyes  and  Whitney  for  the  conditions  of  solution  of  a  sub- 
stance in  water.  Each  particle  of  the  solid  is  to  be  conceived  as 
surrounded  by  an  infinitely  thin  film  of  saturated  solvent,  and 
if  the  general  bulk  of  the  solvent  be  kept  homogeneous  by  proper 
stirring,  the  velocity  of  solution  will  be  proportional  to  the  dif- 


VOL.  1.]  Taylor. — On  Fermentation.  135 

ference  between  the  concentration  of  a  saturated  solution  and  of 
the  particular  saturation  present  in  a  particular  moment,  in 
accordance  with  the  f ormula  £  =  k  (C  —  c),  C  being  the  con- 
centration of  saturation  and  c  that  concentration  actually 
present  in  a  particular  moment.  The  surface  of  the  solid  must 
be  constant.  These  relations  were  confirmed  by  Bruner  and 
Tolloczko  and  lately  by  Brunner. 

If  on  the  other  hand  the  substrate  is  fluid  or  soluble  and  the 
catalysor  solid,  the  relations  will  be,  so  to  speak,  reversed.  The 
reaction  must  be  conceived  to  occur  only  at  the  film  of  contact  of 
the  particle  with  the  solution,  and  the  substrate  must  be  brought 
to  this  film  and  the  products  removed. 

Systematic  authors  like  Ostwald  have  in  general  applied  the 
law  of  mass  action  and  the  van't  Hoff  theory  of  the  order  of  a 
reaction  to  heterogeneous  systems,  the  formulae  being  modified 
to  meet  the  complicated  conditions.  Under  such  circumstances 
the  progress  of  the  reaction  is  held  to  take  place  only  in  the  film 
of  contact  between  the  two  phases,  and  is  there  proportional  to 
the  dimensions  of  the  surface  of  contact ;  but  otherwise  it  follows 
the  general  law  and  is  proportional  to  the  mass  of  the  reacting 
body  or  bodies,  it  being  assumed  that  the  homogeneity  of  the 
general  bulk  of  the  solvent  is  maintained.  Obviously  the  condi- 
tions would  vary,  depending  upon  whether  the  dimensions  of 
the  surface  of  contact  are  constant,  increase  or  diminish.  Before 
the  work  of  Bredig  this  general  interpretation  had  been  almost 
unconsciously  adopted  also  for  catalytic  reactions,  although  the 
facts  that  the  surface  of  contact  in  the  system  is  steadily  de- 
creasing during  the  progress  of  the  reaction,  and  that  the  pro- 
ducts of  the  reaction  are  not  removed,  obviously  render  the  rela- 
tions very  complex.  For  metallic  colloids  the  suspended  particles 
may  be  assumed  to  be  symmetrical,  probably  globular,  and  under 
these  conditions  there  is  a  definite  relation  between  quantity  and 
surface ;  for  ferments,  however,  the  shape  of  the  particles  -is  prob- 
ably amorphous  and  of  no  regular  symmetry,  so  that  here  the 
alteration  in  the  dimensions  of  the  surface  of  contact  with  the 
progress  in  the  reaction  cannot  be  even  surmised. 

Under  the  conception  of  the  nature  of  suspensions  of  the 
so-called  stable  colloids  first  proposed  by  Quincke  and  van  Bern- 


136  University  of  California  Publications.       [PATHOLOGY 

melen  and  since  widely  amplified  by  many  investigators,  such  a 
colloid  suspended  in  water  forms  two  phases : — a  water-poor  and 
a  water-rich  phase,  in  short,  an  aqueous  and  a  colloidal  phase. 
When  a  third  substance  is  dissolved  in  such  a  two-phase  system, 
it  is  distributed  between  the  two.  The  substance  will  be  taken 
up  in  two  ways :  partly  by  adsorption  at  the  film  of  the  colloidal 
phase,  partly  in  solution  in  both  phases.  Now  a  portion  of  the 
substance  that  has  entered  into  the  colloidal  phase  is  irreversibly 
bound,  a  larger  portion  however  may  be  withdrawn.  The  coeffi- 
cient of  distribution  of  the  substance  in  the  two  phases  will  ob- 
viously depend  in  part  upon  the  concentrations  and  in  part 
upon  the  chemical  relations  that  determine  for  different  sub- 
stances the  extent  of  the  irreversible  combination.  If  now  the 
colloid  happens  to  be  a  ferment,  and  the  dissolved  substance  the 
substrate  of  a  fermentation,  it  is  clear  that  the  law  of  mass  action 
cannot  be  applied  to  the  reaction  under  the  simple  assumption 
that  the  velocity  of  the  reaction  is  proportional  to  the  active 
mass  of  the  substrate  and  to  the  dimensions  of  the  film  of  contact. 
In  such  an  experiment,  account  must  be  taken  of  the  factor  of 
the  coefficient  of  distribution,  and  also  of  the  velocity  with  which 
this  distribution  is  effected,  since  with  the  progress  of  the  reac- 
tion, this  would  be  of  influence.  If  both  the  substrate  and  the 
ferment  be  stable  colloids,  the  situation  would  be  only  the  more 
complicated.  The  difference  between  the  stable  colloids,  like 
starch  and  protein,  and  the  unstable  colloids,  like  the  metallic 
suspensions,  must  never  be  overlooked;  and  they  are  associated 
with  such  pronounced  differences  in  physical  behavior  that  we 
are  not  permitted  to  apply  directly  to  the  stable  colloids  the 
results  of  investigations  with  unstable  colloids.  While  therefore 
it  must  be  conceded  that  the  law  of  mass  action  may  not  be  ap- 
plied to  fermentations  in  the  full  theoretical  sense,  the  actual 
question  is :  To  what  extent  do  the  factors  of  the  coefficient  and 
velocity  of  distribution  produce  deviations  in  the  operation  of 
the  law  of  mass  action  ?  This  is  a  question  for  experimentation, 
and  there  is  very  little  data  bearing  upon  it.  It  is  certain  that 
the  relations  are  different  in  different  fermentations. 

Henri2  has  attempted  a  mathematical  characterization  of  this 
point  of  view.     The  process  of  a  fermentation  is  divided  into 


VOL.  l.]  Taylor. — On  Fermentation.  137 

two  g-eneral  parts :  the  movement  of  the  substrate  to  the  film  of 
colloidal  ferment;  and  the  chemical  reaction  at  the  film.  The 
movement  of  the  substrate  to  the  particle  is  again  divided  into 
two  parts :  the  relation  of  the  coefficient  of  distribution,  and  the 
velocity  of  this  distribution.  The  particles  of  ferment  are  looked 
upon  as  stable  colloids;  there  is  in  the  system  an  equilibrium 
between  the  ferment  particles  and  the  inter-particular  fluid,  de- 
pending in  part  upon  the  mass  of  the  fluid,  in  part  upon  the 
properties  of  the  ferment  particles.  The  water  content  of  the 
particles  may  be  increased  or  decreased  by  alterations  in  the 
mass  of  water.  The  particles  of  ferment  effect  adsorption  of 
substances  in  the  solution,  and  this  adsorption  is  in  part  irre- 
versible, in  large  part  however  reversible.  If  to  a  liter  of  a 
colloidal  solution  of  this  sort  we  add  an  amount  a  of  a  soluble 
substance,  the  volume  of  the  colloidal  phase  v  will  be  notably 
altered,  the  volume  of  the  water  phase  V  on  the  contrary  very 
little.  The  substance  added  will  be  divided  between  the  two 
phases  V  and  r.  The  portion  that  has  entered  into  the  colloid 
phase  we  will  term  b;  in  the  water  phase  there  remains  there- 
fore a — b.  The  concentration  in  the  water  phase  therefore 
A 2, 

equals  — =   .=  CV     The  concentration  in  the  colloid  phase  is 

—  C2.     Since    V  -f-  v  —  1,    C2  =   1 y  .     The  portion   of   the 

substance  held  by  the  colloid  b  is  held  partly  in  reversible, 
partly  in  irreversible  absorption.  We  will  term  the  portion  held 
in  reversible  absorption  c,  and  the  portion  held  irreversibly  will 
be  &  —  c.  b  and  c  will  vary  relatively  with  the  nature  of  the 
substance,  the  colloid  and  also  the  medium  of  solution.  The  con- 
centration of  the  substance  a  held  in  reversible  absorption  c 

equals  Cs  =  -j-=    y  .     A  relation  of  equilibrium  must  hold 

between(71  and  Cs.  The  actual  chemical  reaction  at  the  film  of 
contact  is  considered  by  Henri  to  be  proportional  to  the  mass  of 
the  substrate;  the  mass  of  the  substrate  is,  however,  dependent 
on  this  relationship  of  equilibria.  It  is  obvious  that  this  point 
of  view  is  much  more  in -harmony  with  our  knowledge  of  the 
colloid  state  than  the  older  idea  that  the  reaction  under  these 
circumstances  was  proportional  to  the  dimensions  of  the  surface 


138  University  of  California  Publications.       [PATHOLOGY 

Quite  recently  Nernst  has  defined  a  new  point  of  view  in  the 
contemplation  of  the  velocity  of  reactions  in  heterogeneous  sys- 
tems. Nernst  has  amplified  the  principle  of  Noyes  of  the  solu- 
tion velocity  to  include  the  apparent  velocity  of  reaction,  and 
to  define  just  what  an  experimental  velocity  in  a  heterogeneous 
system  really  means.  There  are  obviously  three  main  processes 
in  such  a  reaction :  the  passage  of  substance  through  the  surface 
of  contact  between  the  two  phases,  the  boundary  film ;  the  chem- 
ical reaction  in  one  of  the  two  phases ;  and  diffusion  to  and  away 
from  the  boundary  film.  Nernst  considers  the  first  process  to 
occur  with  such  rapidity  as  to  have  no  influence  upon  the  rela- 
tions. If  the  chemical  reaction  occurs  with  rapidity,  as  Nernst 
believes  to  be  the  usual  condition,  it  is  apparent  that  the  progres- 
sion of  the  reaction  in  time  depends  only  upon  the  velocity  of 
diffusion.  If  the  reaction  be  rapid  as  compared  to  the  diffusion, 
equilibrium  will  exist  at  the  surface  of  contact,  and  if  proper 
mixing  be  provided  for,  the  velocity  of  reaction  represents  simply 
the  velocity  of  diffusion  of  the  substrate  to  the  surface  of  con- 
tact. The  conception  is  probably  best  stated  in  the  words  of 
Nernst : — 

"Many  facts  have  led  to  the  assumption  that  equilibrium  is 
established  with  extraordinary  rapidity  at  the  surface  of  con- 
tact of  two  phases.  Such  a  condition  is  indeed  a  theoretically 
natural  assumption,  because  at  the  surface  of  separation  of  two 
phases,  as  infinitely  approximated  points,  marked  differences  in 
chemical  potential  would  appear,  and  these  would  obviously  pro- 
duce much  chemical  energy  and  lead  to  great  rapidity  of  reac- 
tion. This  means  nothing  more  than  that  in  each  moment  the 
equilibrium  is  established  very  rapidly  in  the  immediate  neigh- 
borhood of  the  surface  of  separation.  If  one  assumes,  what  is 
mathematically  more  probable,  that  the  surface  of  contact  is  not 
a  mathematical  point  but  rather  a  narrow  area  of  transition,  we 
are  nevertheless  still  concerned  with  dimensions  of  the  order  of 
the  sphere  of  activity  of  molecular  potentials;  and  though  we 
can  then  no  longer  speak  of  an  infinite  velocity  of  reaction,  we 
shall  still  be  dealing  with  such  velocities  as  are  for  practical 
purposes  infinite.  When  we  consider  a  chemical  reaction  from 
this  point  of  view,  for  example  the  solution  of  magnesia  in  dilute 


VOL.  l.]  Taylor. — On  Fermentation.  139 

acids,  we  assume  that  the  magnesia  is  in  each  moment  in  equi- 
librium with  the  solution,  that  is,  the  solution  in  immediate  prox- 
imity to  the  magnesia  is  saturated  and  therefore  alkaline.  The 
diffusing  acid  will  be  entirely  neutralized  at  the  surface  of  con- 
tact ;  the  velocity  of  solution  of  the  magnesia  depends  solely  upon 
the  velocity  with  which  the  acid  diffuses  to  the  layer  of  contact 
of  solvent  and  magnesia. 

"In  recent  times  the  van't  Hoff  theory  of  the  order  of  a 
reaction,  that  is,  the  deduction  of  the  number  of  reacting  mole- 
cules from  the  progression  of  a  reaction,  has  been  often  applied 
to  reactions  in  heterogeneous  systems.  When  one  considers  that 
this  theory  rests  upon  the  calculation  of  the  probability  of  the 
kinetic  collision  of  two  or  more  molecules  in  the  gaseous  state  or 
in  dilute  solution,  it  becomes  clear  that  there  is  no  sense  in  its 
application  to  heterogeneous  systems,  or  at  least  that  there  is 
for  such  application  no  theoretical  foundation  extant.  The  above 
mentioned  considerations,  however,  teach  us  that  the  application 
of  the  van't  Hoff  theory  to  heterogeneous  systems  is  not  only 
without  direct  foundation,  but  indeed  entirely  improper,  because 
in  reactions  in  a  heterogeneous  system,  in  so  far  as  the  reactions 
occur  only  at  the  surface  of  contact  of  the  two  phases,  the  veloc- 
ity is  partly  or  entirely  dependent  upon  the  velocity  of  diffusion, 
which  has  no  connection  with  the  order  of  reactions. ' ' 

(That  this  reasoning  need  not  hold  true  in  chemical  reactions 
of  this  type  is  illustrated  by  the  work  of  Haber  on  the  elec- 
trolytic reduction  of  nitro-benzene ;  the  reaction  at  the  surface 
of  the  electrode  was  found  to  be  slow  compared  with  the  velocity 
of  diffusion  to  the  electrode.  As  contrasted  with  the  total  denial 
of  the  application  of  the  kinetic  theory  of  the  order  of  a  reaction 
to  a  heterogeneous  system,  it  may  be  pointed  out  that  Senter  has 
suggested  that  the  Brownian  movements  in  the  particles  of  a 
colloid  may  in  a  sense  be  compared  to  the  molecular  movements 
postulated  in  a  homogeneous  solution  by  the  kinetic  theory.  The 
general  proposition  that  chemical  reactions  must  occur  with  in- 
finite velocity  in  the  film  of  contact  of  two  phases  has  also  been 
denied  on  thermodynamic  grounds  by  Sand,  who  has  adduced  ex- 
perimental illustrations  to  the  contrary.) 


140  University  of  California  Publications.       [PATHOLOGY 

"A  special  instance  of  heterogeneous  chemical  reactions  is 
afforded  by  the  accelerations  of  reactions  by  colloidal  catalysors 
such  as  platinum-asbestos,  Bredig  solutions,  etc.  Since  these  re- 
actions probably  progress  solely  upon  the  surface  of  the  cata- 
lysor,  the  velocity  will  in  no  way  depend  upon  the  mechanism  of 
the  particular  reaction;  if  the  catalysor  maintains  its  integrity 
through  the  course  of  the  reaction  (which  cannot  be  foreseen  in 
advance),  and  carries  through  its  reaction  on  the  surface  of  con- 
tact with  practically  infinite  rapidity,  the  velocity  will  depend 
upon  the  diffusion  of  the  reacting  bodies  to  the  catalysor. ' ' 

"While  it  is  too  early  to  pass  judgment  upon  this  theory,  whose 
fundamental  import  must  be  apparent,  several  general  considera- 
tions may  be  pointed  out.  One  is  that  the  relations  in  fermenta- 
tions will  be  much  more  complex,  because  we  have  here  often  a 
colloidal  substrate  and  a  colloidal  ferment  suspended  in  water, 
instead  of  having  a  heterogenicity  depending  upon  only  one 
member  of  the  system.  Since  in  the  case  of  a  colloidal  substrate 
the  reaction  is  held  to  occur  on  the  surface  of  its  particles,  and 
in  the  case  of  a  colloidal  catalysor  the  reaction  is  held  to  occur 
on  the  surface  of  its  particles,  when  these  conditions  are  united 
in  one  system,  the  reaction  ought  to  be  very  slow;  experience, 
however,  has  taught  us  that  some  of  these  reactions  are  quite 
rapid.  Secondly,  the  Nernst  reasoning  is  based  upon  the  assiimp- 
tion  that  the  solid  body  is  so  slightly  soluble  in  the  medium  of 
the  reaction  that  it  does  not  participate  appreciably  in  the  diffu- 
sion. This  condition  will  in  all  probability  be  found  not  to  hold 
good  for  many  of  the  pseudo-colloidal  substrates  employed  in 
fermentations.  Thirdly,  the  theory  assumes  that  the  catalysor 
is  not  altered  in  the  course  of  the  reaction,  whereas  in  most  fer- 
mentations the  contrary  is  the  case. 

An  argument  against  the  Nernst  theory  lies  in  the  tempera- 
ture coefficient  of  fermentations.  The  velocity  of  chemical  reac- 
tions is  greatly  accelerated  by  increase  in  temperature ;  the  veloc- 
ity of  diffusion  only  slightly.  Now  in  the  case  of  most  ferments 
an  increase  in  temperature  is  followed  by  the  increase  in  velocity 
that  would  be  expected  in  a  chemical  reaction — it  is  usually  more 
than  doubled  in  10  degrees — far  more  than  would  be  expected 
were  a  process  of  diffusion  alone  concerned. 


VOL.  1.]  Taylor. — On  Fermentation.  .  141 

Another  objection  is  that  proportional  increases  in  viscosity 
in  the  fermenting  system,  due  to  the  addition  of  different  sub-' 
stances,  ought  to  exert  proportional  retardations  of  the  veloc- 
ity, if  that  velocity  express  simply  the  diffusion  velocity.  This 
is  not  the  case.  Similarly,  in  heterogeneous  reactions  of  known 
chemical  nature,  variations  in  concentration  and  viscosity  ought 
to  produce  the  same  variations  in  velocity  as  in  fermentation 
experiments ;  but  this  has  not  been  observed  to  be  true. 

Lastly  it  must  be  pointed  out  that  the  term  colloid  does  not 
correspond  to  a  fixed  quality,  but  to  a  tendency  to  physical  quali- 
ties that  is  more  or  less  pronounced  in  different  substances.  For 
Graham  the  colloid  was  the  non-diffusible  body,  the  crystalloid 
the  diffusible  body.  Colloids  confer  upon  their  solutions  or  sus- 
pensions a  very  slight  depression  of  the  freezing  point  or  eleva- 
tion of  the  boiling  point,  and  possess  a  very  low  osmotic  pressure, 
all  of  which  indicate  that  the  work  necessary  to  effect  the  sepa- 
ration of  the  colloid  from  the  medium  is  small ;  with  crystalloids 
the  contrary  is  true.  Like  all  suspensions,  colloidal  solutions 
display  peculiar  conditions  of  precipitation  and  coagulation,  and 
are  very  active  in  all  that  relates  to  surface  tension  and  adsorp- 
tion. But  it  is  not  at  all  true  that  all  chemical  bodies  belong  to 
one  or  the  other  of  these  classes.  On  the  contrary,  as  indicated 
by  Spring  and  Lobry  de  Brun,  there  are  innumerable  interme- 
diary conditions,  corresponding  to  all  conceivable  gradations 
from  the  typical  crystalloid  to  the  typical  colloid.  Not  only  this, 
many  substances  display  attributes  quite  extreme;  thus  prota- 
mines  will  not  crystallize,  but  will  diffuse  well  and  will  transport 
an  electrical  current,  while  some  higher  proteins  will  crystallize 
and  yet  not  diffuse  with  measurable  rapidity;  and  while  some 
are  quite  insoluble  in  the  true  sense,  others  are  quite  soluble. 
Under  these  circumstances,  dealing  with  bodies  displaying  all 
degrees  of  gradation  from  the  typical  crystalloid  to  the  typical 
colloid,  it  is  difficult  to  foresee  to  what  extent  the  theory  of 
Nernst,  based  upon  the  qualities  of  practically  pure  heteroge- 
nicity  in  the  system,  will  bear  the  test  of  experimental  applica- 
tion to  fermentations.  The  colloids  with  which  we  are  dealing  in 
fermentations  are  in  many  respects  different  from  the  metallic 
suspensions  to  which  the  theory  finds  direct  application.  They 


142  University  of  California  Publications.       [PATHOLOGY 

react  differently  to  electrolytes,  resist  precipitation  by  them,  are 
indeed  in  some  instances  protected  from  precipitation  by  them, 
and  they  have  the  power  to  protect  genuine  colloids  from  pre- 
cipitation by  electrolytes.  Under  ultramicroscopic  examination, 
the  stable  (or  pseudo)  colloids  are  seen  to  possess  much  smaller 
particles  than  the  true  colloids;  and  corresponding  to  this  they 
display  crystalloid  tendencies — power  of  diffusion,  osmotic  pres- 
sure, etc.  In  fact,  some  of  the  proteins  have  as  pronounced 
crystalloidal  properties  as  many  dye-stuffs  (which  are  commonly 
considered  as  real  crystalloids),  and  scarcely  more  measurable 
colloidal  properties.  Obviously  the  theory  of  Nernst  cannot 
be  applied  to  such  bodies  with  complete  'theoretical  validity. 
That  the  conditions  upon  which  this  theory  is  based  will  often  be 
of  great  influence  in  the  progressions  of  reactions  must  be  con- 
ceded in  advance;  it  is  indeed  a  priori  probable  that  in  many 
instances  the  experimental  velocity  will  be  the  result  of  both  a 
true  reaction  velocity  and  the  diffusion  velocity;  but  whether  it 
will  afford  in  itself  an  adequate  explanation  of  all  the  phenom- 
ena is  very  much  to  be  doubted  so  far  as  biological  fermentations 
are  concerned.  As  will  be  illustrated  later,  the  results  in  many 
instances  correspond  very  closely  with  the  requirements  of  the 
older  law.  The  investigations  of  Heimbrodt  indicate  that  with 
constant  conditions  of  diffusion,  the  differential  equation  de- 
manded by  the  Nernst  theory  is  identical  with  that  for  a  mono- 
molecular  reaction.  In  other  instances,  however,  such  conform- 
ity has  not  been  obtained,  and  it  may  be  in  precisely  these  cases 
that  the  conditions  elucidated  by  Nernst  have  played  predomi- 
nating roles.  The  cleavage  of  an  insoluble  fat  by  lipase  repre- 
sents, as  will  be  later  noted  in  detail,  a  probable  illustration  of  a 
diffusion  velocity. 

Another  factor  which  must  modify  the  relations  in  the  com- 
plex fermentations  of  biological  order  is  the  relations  of  colloids 
to  each  other.  The  adsorption  of  stable  colloids  by  one  another 
is  not  a  function  of  surface  tension ;  it  is  in  part  at  least  a  func- 
tion of  composition.  The  adsorption  of  a  particular  colloid  by 
two  different  colloids  even  of  the  same  surface  tension  is  not  the 
same.  It  is  the  usual  teaching  that  colloids  cannot  diffuse,  but 
the  phenomena  of  adsorption  throw  grave  doubt  upon  this  state- 


VOL.  l.]  Taylor. — On  Fermentation.  143 

merit.  Colloidal  hydrosols  will  penetrate  hydrogels  in  a  progres- 
sive linear  manner.  That  this  may  not  be  a  true  diffusion,  but 
rather  a  co-solution,  a  relation  of  the  coefficient  of  distribution, 
possibly  even  a  chemical  combination  with  the  formation  of  a 
new  colloidal  complex,  is  freely  granted.  But  the  fact  will  no 
less  indicate  that  the  phenomenon  under  whatever  name  it  pass 
must  constitute  a  variable  in  the  reactions  in  such  a  system. 

In  the  concrete  application  of  the  Nernst  theory  to  fermenta- 
tions, as  proposed  by  Herzog,  it  is  assumed  that  the  diffusion 
velocity  is  the  function  of  the  viscosity  of  the  medium,  and  the 
equation  is  erected  on  magnitudes  derived  from  this  considera- 
tion. The  work  of  Herzog  will  be  discussed  in  detail  under  in- 
version. 

It  is  necessary  further  to  consider  the  nature  of  the  relations 
between  the  substrate  and  the  ferment.  We  know  on  the  one 
hand  that  colloids  enter  into  complex  chemical  combinations  with 
other  substances.  We  have  evidence  that  complex  combinations 
of  this  sort  occur  in  organisms:  such  are  the  sugar-protein,  the 
protein-fat,  the  purin-nucleine,  the  lecethin-protein  complexes. 
These  seem  destined  to  play  an  important  role  in  the  chemical 
physiology  of  the  future.  As  chemical  combinations  these  com- 
plexes are  subject  to  the  laws  of  mass  action,  equilibrium  and 
partition.  On  the  other  hand,  we  know  that  colloids  form  with 
other  substances  physical  equilibria  that  van  Bemmelen  has 
termed  adsorption  compounds.  Recent  investigations  seem  to 
indicate  that  for  the  typical  unstable  colloids  (metallic  hydro- 
sols, silicate,  etc.)  the  laws  of  mass  action,  equilibrium  and  par- 
tition do  not  seem  to  hold.  For  the  atypical  colloids  (protein, 
starch,  etc.)  these  laws  seem  to  tend  to  hold.  It  is  fairly  certain 
that  for  some  reactions  (e.g.,  the  system  dye-cellulose)  the  early 
occurrence  of  secondary  reactions  affecting  the  one  component 
(the  dye)  are  responsible  for  the  non-fulfillment  of  these  laws 
and  the  cause  of  the  irreversibility  of  the  reaction;  such  a  phe- 
nomenon, however,  does  not  constitute  a  fundamental  exception 
to  the  laws.  Concerning  these  matters  we  possess  as  yet  so  little 
quantitative  data  that  definite  conclusions  are  not  warranted. 
Nevertheless,  unless  we  can  conceive  that  when  two  substances 
meet  in  a  thin  film  on  the  surface  of  a  third  indifferent  sub- 


144  University  of  California  Publications.       [PATHOLOGY 

stance  they  react  with  infinite  velocity,  we  shall  be  compelled  to 
consider  the  combination  of  ferment-substrate  to  be  of  the  nature 
of  a  chemical  complex  rather  than  of  a  physical  adsorption  com- 
pound. 

In  its  broadest  application,  the  fact  upon  which  the  theory  of 
Nernst  is  founded,  that  reactions  occur  with  great  velocity  at 
the  boundary  of  contact  of  two  phases,  must  appear  to  every  one 
as  a  fact  of  the  deepest  biological  significance.  When  we  con- 
sider that  the  cells  of  the  body  in  their  relations  to  the  circulat- 
ing fluids,  indeed  the  fibrillar  and  granular  parts  of  cells  in  their 
relations  to  the  intercellular  fluids,  represent  precisely  just  such 
two-phase  systems,  we  realize  the  magnitude  of  the  factor  with 
which  we  are  dealing.  From  the  physical  point  of  view,  the  cel- 
ular  constructions  are  simply  colloidal  phases,  hydrogels,  the 
body  fluids  watery  phases,  hydrosols.  The  magnitude  of  the 
dimensions  of  the  boundaries  of  contact  of  the  two  phases  in  a 
human  body  is  almost  inconceivable ;  and  the  influence  upon  the 
velocity  of  any  reaction  under  these  circumstances  as  contrasted 
with  the  velocity  of  the  same  reaction  in  a  simple  system  of  the 
same  bulk  must  be  enormous.  Though  it  is  a  pure  speculation,  I 
cannot  refrain  from  expressing  the  thought  that  to  this  factor 
may  be  ascribed,  in  part  at  least,  the  great  difference  in  velocity 
everywhere  to  be  observed  between  reactions  in  vivo  and  in  vitro. 

Very  recently  (in  fact,  since  these  lectures  were  delivered) 
Henri3  has  advanced  some  general  ideas  of  ferment  action  which 
combine  the  chemical  point  of  view  of  the  reaction  velocity  with 
the  physical  properties  of  a  colloidal  system.  His  views  may  be 
summarized  as  follows :  Soluble  ferments  are  solutions  of  stable 
colloids.  Colloids  are  heterogeneous  media  formed  of  granules 
suspended  in  an  intergranicular  fluid.  Stable  colloids  are  formed 
of  granules  rich  in  water.  Between  the  granules  and  the  inter- 
granicular fluid  a  state  of  equilibrium  exists ;  the  composition  of 
the  granules  has  a  direct  relation  to  that  of  the  liquid.  In  the 
variation  of  the  composition  of  the  granules  one  must  distinguish 
two  factors :  variation  in  the  water  content,  and  variation  in  the 
adsorption,  by  the  granules,  of  substance  dissolved  in  the  inter- 
granicular fluid.  Substance  adsorbed  by  the  granules  is  in  two 
forms:  that  adsorbed  irreversibly,  and  that  held  in  reversible 


V°L-  !•]  Taylor. — On  Fermentation.  145 

combination.  The  division  of  the  solute  between  the  granules 
and  the  intergranicular  fluid  follows  a  law  of  coefficient  of  dis- 
tribution. The  essential  character  of  the  action  of  ferments  lies 
in  a  relation  between  the  reaction  velocity  and  the 'concentration 
of  the  solution;  this  relation,  however,  depends  upon  the  coeffi- 
cient of  distribution  of  the  substance  between  the. granules  and 
the  intergranicular  fluid. 

Reactions  in  a  heterogeneous  system  may  be  divided  into 
t\vo  groups:  hetereogeneous  catalysis,  and  reactions  between 
substances  in  twro  phases.  In  each  of  these  the  reaction  may 
be  conceived  to  occur  on  the  surface  of  separation  of  the  two 
phases  or  in  the  interior  of  one  of  the  phases.  The  velocity 
of  reaction  in  a  heterogeneous  system  will  depend  upon  the 
following  factors:  (a)  The  velocity  of  the  chemical  reaction 
%)er_se,  which  shall  be  termed  R.  (b)  The  velocity  with  which 
the  bodies  that  react  arrive  at  the  boundary  of  contact  of  the 
two-  phases,  or  penetrate  into  the  interior  of  the  phase  where 
the  reaction  occurs ;  this  velocity  is  determined  directly  to  be  the 
coefficient  of  diffusion-jctf  the  reacting  body  D.  (c)  The  concen- 
tration of  the  reacting  bodies  at  the  site  of  reaction ;  if  the  reac- 
tion occur  in  the  interior  of  one  of  the  phases,  this  will  be  ex- 
pressed in  the  coefficient  of  distribution,  (d)  The  jjiinensions  of 
the  ^surface  of  contact  of  the  two  phases^,  ajid  the  volume  of 
the  phase  in  which  the  reaction  occurs  V.  I.  If  R  be  very 
rapid,  the  reaction  will  occur  solely  at  the  boundary  of  contact 
of  the  two  phases,  the  law  of  distribution  is  eliminated,  the  film 
of  separation  of  the  two  phases  has  a  constant  thickness  and  com- 
position, the  velocity  of  diffusion  alone  is  the  operative  factor. 
(This  is  the  hypothesis  of  Nernst.)  II.  If  R  be  relatively  slow, 
D  and  the  distribution  very  rapid,  the  reaction  will  occur  in  the 
interior  of  one  of  the  phases,  and  the  reaction  velocity  will  de- 
pend on  the  concentration  of  the  reacting  bodies,  if  8  and  V  are 
constant.  III.  If  R  is  rapid,  and  8  is  variable,  the  velocity  of 
the  reaction  occurring  at  the  film  of  contact  of  the  two  phases 
will  depend  on  the  diffusion  and  the  dimensions  of  the  surface  8. 
Henri  considers  that  in  those  fermentations  in  which  the  sub- 
strate is  a  crystalloid  and  the  ferment  a  colloid,  the  phenomena 
are  to  be  ranged  under  II  or  III.  In  the  case  of  those  fermenta- 


146  University  of  California  Publications.       [PATHOLOGY 

tions  in  which  both  the  substrate  and  the  ferment  are  colloids, 
the  relations  will  be  very  much  complex,  but  they  may  also  be 
ranged  under  II  or  III.  Obviously  the  possible  combinations  of 
these  several  factors  are  not  exhausted  by  the  I,  II,  and  III  of 
Henri.  On  the  assumption  that  the  actual  reaction  is  propor- 
tional to  the  masses  of  the  reacting  bodies  and  is  further  a  func- 
tion of  the  partition  and  equilibrium  between  the  solution  and 
the  colloid  phase,  Henri  has  developed  a  general  equation  theo- 
retically applicable  to  all  fermentations.  Henri  has  as  yet  pub- 
lished no  experimental  data  bearing  directly  on  the  classification 
of  particular  fermentations  under  these  headings.  That  the 
scheme  includes  all  the  possible  factors  involved  in  this  phenom- 
enon may  be  granted.  To  what  extent  these  several  factors  will 
be  found  to  be  operative  in  their  relations  to  each  other  in  partic- 
ular fermentations  cannot  be  foreseen.  That  difficulties  will  ap- 
pear in  the  formulation  of  the  problem  and  especially  in  the 
different  measurements  is  equally  obvious.  Nevertheless*  the 
scheme  is  very  lucid,  and  attractive  in  that  it  conserves  the 
chemical  nature  of  the  phenomenon  and  locates  the  adventitious 
variables  that  lie  outside  of  it  in  the  domain  of  the  physical  prop- 
erties of  colloids,  as  defined  in  one  of  the  most  current  and  best 
elucidated  theories  of  the  colloid  state.  The  weakness  of  the 
scheme  lies  in  the  disregard  of  the  fact  that  many  of  the  so-called 
colloids  with  which  we  are  dealing  are  more  or  less  atypical,  and 
deviate  greatly  from  the  typical  colloid  in  the  direction  of  crystal- 
loidal  properties.  It  has  become  the  fashion  in  biological  circles 
to  attribute  the  most  numerous  and  diverse  of  unexplained  phe- 
nomena to  the  physical  properties  of  the  colloidal  state.  Valuable 
as  the  physical  point  of  view  unquestionably  is,  in  the  investiga- 
tion of  the  concrete  problem  such  general  assumptions  must  be 
replaced  by  experimental  work. 


VOL.  1]  Taylor — On  Fermentation.  147 


LITERATURE. 

Abelous  &  Gerard.     C.  r.  Acad.  Sc.,  129-50,  164,  1023. 

Armstrong.    Proc.  Koy.  Soc.,  74-511. 

Baeyer.     Berichte,  5-68. 

Bertrand.1    C.  r.  Acad.  Sc.,  118-1215;  120-266;  121-166,  783;  122-900,  1132, 

1215;  125-463. 

Bertrand.2    C.  r.  Acad.  Sc.,  126-702,  842,  984;  127-124,  728. 
Bredig.1    Die  Anorgan.  Fermente. 
Bredig.2    Ergebnisse  der  Physiologie,  1-191. 
Erode.     Zeitschr.  f.  physik.  Chem.,  57-257. 
Bruner  &  Tolloczko.     Zeitschr.  f.  physik.  Chem.,  55-283. 

Zeitschr.  f.  anorgan.  Chem.,  28-314;  35-23. 
Brunner.    Zeitschr.  f.  physik.  Chem.,  47-56. 
Burian.    Zeitschr.  f.  physiol.  Chem.,  43-497. 
Engler  &  Weissberg.     Krit.  Stud.  u.  d.  Vorg.  d.  Autoxydation. 
Ernst.     Zeitschr.  f.  physik.  Chem.,  57-448. 
Erlenmeyer.     Berichte,  1 0-634. 
Euler.1    Berichte,  37-3411. 
Euler.2    Berichte,  38-2551. 
Fawsitt.    Zeitschr.  f.  physik.  Chem.,  41-601. 
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Rev.  d'Igiene,  1892,  201. 
Heimbrodt.    Ann.  d.  Physik.  (IV),  15-1028. 
Henri.1    Lois  generates  de  1 'Action  des  Diastases,  85. 
Henri.2    C.  r.  Soc.  Biol.,  55-612. 

Zeitschr.  f.  physik.  Chem.,  51-19. 
Henri.3    Zeitschr.  f.  Electrochem.,  11,  790. 
Hoppe-Seyier.1    Arch,  f .  d.  ges.  Physiol.,  12-11. 
Hoppe-Seyler.2     Zeitschr.  f.  physiol.  Chem.,  2-13. 
Jacquet.     Arch.  f.  exper.  Path.  u.  Phar.,  #9-386. 
Jahn.     Zeitschr.  f.  physik.  Chem.,  50-129. 
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Knoblauch.    Zeitschr.  f.  physik.  Chem.,  22-268. 
Koelichen.     Zeitschr.  f.  physik.  Chem.,  55-129. 
Kossel.     Zeitschr.  f.  physiol.  Chem.,  41-321;  42-181. 
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Maclntosch.     Jour.  Physic.  Chem.,  6-15. 
Xernst.     Zeitsehr.  f.  physik.  Chem.,  47-52. 


148  University  of  California  Publications.      [PATHOLOGY 

Noyes.     Zeitsehr.  f.  physik.  Chem.,  19-599. 
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Tammann.     Zeitsehr.  f.  physik.  Chem.,  3-25;  18-426. 

Zeitsehr.  f.  physiol.  Chem.,  16-271. 
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VOL.  1]  Taylor — On  Fermentation.  149 


FERMENTATION  OF  CARBOHYDRATES. 

In  the  fermentation  of  carbohydrates  we  have  to  deal  with 
three  general  groups  of  ferments.  The  ferments  of  one  group 
accelerate  the  hydrolysis  of  the  polysaccharides  to  di-  or  mono- 
saccharides.  Starches  are  as  a  rule  hydrolyzed  only  to  di-sac- 
cha rides,  which  then  fall  into  the  second  division.  There  is,  on 
the  contrary,  some  evidence  tending  to  indicate  that  cellulose  may 
be  hydrolyzed  directly  to  mono-saccharides,  though  it  has  not 
been  demonstrated  that  but  one  ferment  is  concerned.  The  fer- 
ments of  the  second  group  accelerate  the  inversion  of  disaccha- 
rides  to  mono-saccharides.  These  ferments  often  accompany 
those  of  the  ferments  of  the  first  group,  so  that  their  actions  are 
superimposed.  The  ferments  of  the  third  group  accelerate  the 
conversion  of  mono-saccharides  into  substances  like  alcohol,  acids, 
etc. 

Strictly  speaking,  as  Fischer  has  insisted,  the  glucosides 
should  be  classed  with  the  di-saccharides  rather  than  with  the 
starches;  they  consist  of  combinations  of  primary  sugars  with 
organic  acids,  alcohols,  aldehydes,  or  aromatic  substances;  and 
though  they  often  contain  more  than  one  molecule  of  sugar,  it  is 
usually  the  same  sugar,  and  the  two  molecules  are  apparently 
combined  with  the  other  component  rather  than  with  each  other. 
Their  ferments  furthermore  are  of  the  type  of  invertase.  It  has 
been  observed  that  other  ferments  coexist  with  them,  and  the  re- 
sults were  superimposed,  and  this  led  to  a  great  deal  of  confusion 
in  the  early  literature. 

We  shall  limit  ourselves  to  the  consideration  of  the  relations 
of  six  of  these  ferments:  amylase  (fermentation  of  starch),  iii- 
i'ermeiitation  of  cane  sugar),  maltase  (fermentation  of 
malt  sugar),  laccase  (fermentation  of  milk  sugar),  emulsin  (fer- 
mentation of  glucosides),  and  zymase  (fermentation  of  glu- 
cose). Not  only  are  these  the  most  prominent  members  of  their 
series,  but  they  have  been  studied  in  the  most  thorough  manner. 


150  University  of  California  Publications.      [PATHOLOGY 


FERMENTATION  OF  STARCH. 

The  fermentation  of  starch  shares  with  alcoholic  fermenta- 
tion the  greatest  age  of  investigation.  Kirchoff,  who  discovered 
the  acid  hydrolysis  of  starch,  first  described  the  conversion  of 
starch  into  sugar  through  the  agency  of  fresh  gluten.  Dubrun- 
faut  showed  that  a  particular  substance  in  the  grain  possessed 
this  activity,  was  present  to  greatest  degree  during  germination, 
was  soluble  in  water,  and  that  the  sugar  produced  was  not  iden- 
tical with  that  obtained  through  the  action  of  acids  upon  starch. 
Payen  separated  the  raw  ferment  from  its  solutions  by  precipi- 
tation with  alcohol.  Since  that  time  the  action  of  amylase  has 
been  studied  to  an  enormous  extent,  in  large  part  by  the  technical 
chemists  engaged  in  the  fabrication  of  alcohol  and  maltose,  and 
in  the  brewing  of  beer.  The  presence  of  amylytic  ferment  in  the 
saliva  was  discovered  by  Leuchs,  in  the  pancreatic  secretion  by 
Bouchardat  and  Soudras,  and  in  the  succus  entreicus  by  Roehr- 
mann.  It  is  present  also  in  the  liver,  in  muscular  tissue,  and  in 
the  urine.  Whether  the  traces  that  may  be  found  in  the  other 
organs  and  tissues  of  the  body  represent  local  formation  or  sim- 
ply the  deposition  from  the  blood  is  not  known.  The  presence 
of  amylase  in  the  blood  is  probably  a  secondary  condition,  de- 
pendent upon  the  liver. 

Amylase  is  very  widely  distributed.  In  addition  to  being 
found  in  all  higher  species,  it  is  known  to  be  present  in  insects, 
arthropodae,  sponges,  shell  fish,  coelenterae,  echinoderms,  and 
even  in  protozoa.  It  has  been  described  in  the  eggs  of  some  crus- 
taceans. In  all  animals  there  are  apparently  two  locations  of 
the  ferment:  one  in  the  secretions  of  the  digestive  glands,  and 
the  other  in  the  liver.  In  the  vegetable  kingdom  its  distribution 
is  equally  wide.  It  is  apparently  present  in  seeds  during  the 
period  of  germination,  and  in  many  grains  to  some  extent  during 
the  resting  period.  It  is  also  present  in  trunks,  stalks,  bulbs,  in 
sprouts  of  all  kinds,  and  in  leaves  of  plants  of  all  degrees  of 
dignity.  It  is  also  present  in  many  fungi,  such  as  different  va- 
rieties of  aspergilli,  sacchromyces,  in  all  the  common  yeasts,  in 
the  bacteria  that  ferment  wood,  and  in  many  bacteria  and  cocci, 


VOL.  i]  Taylor — On  Fermentation.  151 

especially  in  the  cholera  vibrio,  in  the  group  bacterium  termo, 
the  proteus  vulgaris,  and  the  bacillus  mesentericus  vulgatus. 
The  amylytic  activity  is,  however,  not  a  prominent  faculty  of 
disease-producing  germs  in  general,  though  it  seems  to  be  a  com- 
mon faculty. 

Whether  in  all  these  situations  we  are  dealing  with  the  same 
ferment  can  only  be  conjectured.  There  is  on  the  one  hand  no 
reason  why  the  acceleration  of  the  hydrolysis  of  starch  should 
be  limited  to  one  substance,  and  it  is  not  natural  to  suppose  that 
all  these  different  plants  and  animals  should  be  so  united  upon 
this  one  point  when  they  vary  so  widely  in  other  functions. 
And.  furthermore,  the  diastatic  ferments  differ  so  widely  in  their 
relations  to  different  temperatures  and  to  the  action  of  various 
chemical  substances  that  this  affords  concrete  grounds  for  at- 
tempts at  differentiation.  At  the  same  time,  recent  studies  in 
colloids  have  indicated  that  different  preparations  of  a  colloidal 
metal  of  chemical  purity  will  exhibit  widely  different  properties, 
depending  upon  the  circumstances  of  preparation,  conservation, 
etc.,  so  that  from  such  differences  alone  separate  chemical  entities 
cannot  be  inferred.  Kjeldahl  has  shown  that  when  a  particular 
preparation  of  amylase  is  exposed  for  some  time  to  a  higher  tem- 
perature, it  will  exhibit  differences  in  behavior  of  surprising  ex- 
tent as  compared  with  the  qualities  of  the  parent  stock,  and  in 
many  instances  these  artificial  differences  are  greater  than  those 
observed  in  different  preparations  and  relied  upon  to  indicate  a 
multiplicity  of  ferments.  As  a  matter  of  actual  fact,  we  possess 
so  little  definite  knowledge  upon  the  matter,  knowledge  based 
upon  study  of  pure  preparations,  that  a  definite  opinion  is  not 
warranted. 

The  same  considerations  hold  true  with  reference  to  the  indi- 
viduality of  any  particular  amylase.  The  reaction  consists  of 
two  separate  functions,  the  liquefaction  and  the  saccharification 
of  starch.  The  liquefaction  of  starch  means  the  conversion  of  a 
hydrogel  into  a  hydrosol,  and  it  is  difficult  to  consider  this  re- 
lated to  the  hydrolysis  of  the  starch.  Not  only  are  the  proced- 
ures different,  but  the  relations  of  temperature  and  of  extraneous 
substances  in  the  solvent  are  different  for  the  two  procedures. 
According  to  the  interpretation  of  Maquenne  and  Roux.1  starch 


152  University  of  California  Publications.      [PATHOLOGY 

consists  of  two  substances,  amylo-cellulose  and  amylo-pectin.  The 
liquefaction  ferment  acts  on  the  amylo-pectin,  the  saccharifica- 
tion  ferment  acts  on  the  amylo-cellulose.  Fernbach  and  Wolff1 
have  described  preparations  of  amylase  that  had  no  power  of 
liquefaction,  but  were  active  in  saccharification.  It  has  been 
natural  therefore  to  assume  that  amylase  contains  two  different 
ferments.  If  we  judge  from  the  history  of  ferments,  this  will 
probably  prove  to  be  true,  but  it  has  not  been  demonstrated 
directly.  Furthermore,  it  has  been  long  claimed  that  the  hydro- 
lytic  acceleration  is  due  to  two  rather  than  to  one  ferment ;  the 
one  being  supposed  to  accelerate  the  conversion  of  starch  into 
dextrines,  the  second  to  accelerate  the  conversion  of  the  dextrines 
into  maltose.  In  the  present  state  of  our  knowledge,  this  hypoth- 
esis can  be  neither  affirmed  nor  denied.  One  must,  however,  be 
very  careful  in  grasping  at  the  assumption  of  a  different  ferment 
for  each  sub-stage  of  a  reaction,  otherwise  we  shall  be  soon  pos- 
sessed of  as  many  hypothetical  ferments  as  we  are  supposed  to  be 
possessed  of  anti-bodies. 

Wherever  found,  amylase  is  apparently  a  secretion  product 
of  cells.  It  is  secreted  in  an  inactive  form,  as  a  zymogen,  which 
on  suspension  in  water  becomes  converted  into  the  active  fer- 
ment. The  quantitative  relations  of  this  activation,  as  well  as 
the  fact  that  it  may  be  accelerated  by  many  substances,  leads  to 
the  inference  that  the  process  of  activation  is  in  reality  an  act  of 
hydrolysis,  thus :  amylase-zymogen  -f-  water  =  amylase.  Some 
botanists  have  attempted  to  separate  two  forms  of  amylase,  the 
secretion  and  the  translocation  ferment;  a  chemical  differentia- 
tion has  not  been  demonstrated.  The  sum  total  of  our  knowledge 
of  the  relations  in  plants  suggests  that  not  only  does  amylase 
accelerate  the  formation  of  maltose  from  starch,  it  also  acceler- 
ates the  formation  of  starch  from  maltose. 

TJie  Hydrolysis  of  Starch.  This  has  been  a  bone  of  great  con- 
troversy. That  the  end  product  is  maltose  is  undoubted.  The 
reaction  runs: 

Starch  +  water  —  maltose  +  maltose. 

n   (Cl2H20010)n    +nH,0  =  (C12H«Ou)n+  (CHH«On)D. 

The  reaction  under  favorable  conditions  may  be  complete  in 
the  sense  that  the  starch  has  disappeared,  but  it  is  incomplete 


VOL.  l]  Taylor — On  Fermentation.  153 

in  the  sense  that  the  substance  has  not  all  been  converted  into 
maltose.  Lea  has  shown  that  if  the  products  of  the  reaction  be 
removed  from  the  system,  the  reaction  will  be  completed.  The 
reaction  has  many  sub-stages;  it  is  a  mediated  catalysis.  In  the 
acid  catalysis  of  starch,  similar  intermediary  stages  appear,  with 
the  formation  of  successive  dextrinous  bodies.  The  reactions  of 
these  dextrines  are  very  indefinite,  and  one  cannot  read  the  work 
of  Musculus,  Lintner  &  Duell,  Brown  and  Morris,  Bourquelot, 
Duclaux,  and  others  without  becoming  convinced  firstly  that  the 
number  of  sub-stages  in  the  reaction  is  not  known,  and  secondly 
that  for  those  intermediary  stages  that  seem  quite  definite,  the 
corresponding  products  offer  at  present  no  definite  distinguishing 
properties.  It  is  difficult  to  secure  an  inversion  of  starch  without 
the  persistence  of  some  of  these  dextrinous  substances. 

Concerning  the  actual  steps  in  the  hydrolytic  cleavage  of 
starch,  there  are  divergent  opinions.  Here,  as  elsewhere,  the' 
less  definite  the  experimental  data,  the  more  uncontrolled  the 
inductions.  Based  upon  the  fact  that  the  cleavage  is  never  com- 
plete, dextrine  always  remaining,  is  the  hypothesis  that  the  reac- 
tion might  be  a  direct  cleavage : 

Starch  +  water  =  dextrine  +  maltose. 

This  hypothesis  is  not  in  accord  with  the  investigations  in  frac- 
tional fermentation,  and  the  fact  of  dextrine  remaining  with  the 
end  products  is  more  easily  interpreted  on  the  basis  of  a  limited 
reaction. 

A  second  hypothesis  is  that  successive  dextrines  are  formed, 
and  from  the  final  dextrine  maltose  is  formed. 

Starch  +  water  =  dextrine!. 
Dextrine!  +  water  =  dextrine,. 
Dextrinen  +  water  =  maltose  +  maltose. 

The  third  hypothesis  is  that  molecules  of  maltose  are  succes- 
sively split  of  the  successive  dextrines. 

Starch  +  water  =  dextrine!  +  maltose. 
Dextrine!  +  water  =  dextrine2  +  maltose. 
Dextrinen  +  water  =  maltose  +  maltose. 

As  between  these  two  hypotheses,  the  available  data  yield  no 
decision.  From  the  point  of  view  of  the  investigation  of  the 


154  University  of  California  Publications.      [PATHOLOGY 

dynamics  of  the  process,  neither  would  be  preferable  over  the 
other,  since  in  both  we  have  a  series  of  reactions  of  which  we  can 
measure  only  the  disappearance  of  the  starch  and  the  appearance 
of  the  maltose.  No  method  of  measuring  the  conversion  of  starch 
into  the  first  dextrine  has  been  established ;  and  the  measurement 
of  the  maltose  is  rendered  uncertain  until  we  learn  that  the  lower 
dextrines  do  not  reduce  Fehling's  solution  or  disturb  its  regular 
reduction  by  maltose. 

According  to  the  latest  investigations  of  Moreau,  the  reactions 
comprise  a  combination  of  the  last  two  schemes.  He  gives  the 
following  scheme : 

STARCH. 


I  I  I 

Achroodextrine.     Erythrodextrine.       Amylodextrine.       Maltose. 


Maltose.     Erythrodextrine.    Achrodextrine. 


I 
Achroodextriue.        Maltose. 


Maltose. 


Maltose.  Maltose.        Achroodextrine.         Maltose.  Residue. 

Maltose. 

Whether  oxygen  is  necessary  to  the  action  of  amylase  has 
been  often  discussed.  Recent  experiments  of  Sacharow  would 
seem  to  indicate  that  it  is  necessary. 

The  reaction  of  the  cleavage  of  glycogen  is,  so  far  as  known, 
very  similar  to  that  of  starch.  The  intermediary  dextrines  seem 
on  the  basis  of  color  reactions  less  numerous  than  in  the  case  of 
starch,  and  the  reaction  usually  proceeds  with  greater  rapidity. 
When  a  pure  amylase  is  employed,  the  product  is  maltose.  That 
glucose  is  found  when  mammalian  amylase  is  used  is  due  to 
the  fact  that  an  admixture  of  maltase  is  always  present.  Phi- 
loche1  states  that  with  the  use  of  pancreatic  amylase  the  digestion 
of  glycogen  is  very  like  that  of  soluble  starch.  With  the  use  of 
vegetable  amylase,  on  the  contrary,  the  glycogen  is  much  more 
resistant.  The  curve  of  reaction  rose  rapidly,  soon  to  become 
almost  stationary.  Experiments  at  this  stage  indicated  that  the 


VOL.  1]  Taylor — On  Fermentation.  155 

ferment  was  unaltered,  but  that  the  unconverted  glycogen  no 
longer  gave  the  iodine  reaction,  although  it  was  still  perceptible 
by  alcohol. 

For  the  investigation  of  an  amylase  a  soluble  starch  should 
be  used.  There  are  several  reasons  why  a  raw  starch  is  not  suit- 
able. The  reactions  of  liquefaction  and  sacchqrifi cation  are  su- 
perimposed, and  as  they  are  fundamentally  rliffprpnf.  things,  the 
essential  conditions  of  a  fermentation  experiment  are  not  ful- 
filled. The  rapidity  of  the  action  of  an  acid  or  a  ferment  is 
dependent  to  a  large  extent  upon  the  physical  state  of  the  starch : 
its  fineness  of  subdivision,  its  coating  with  cellulose,  the  lipoids, 
its  dessication.  A  starch  solution  should  be  weak,  from  one- 
fourth  to  one-half  per  cent.  It  must  be  tested  for  its  sugar  con- 
tent. If  this  be  large,  it  must  be  removed  by  dyalization,  not  a 
rapid  procedure  with  maltose ;  if  it  be  low,  it  may  be  neglected. 
The  solution  should  be  opalescent ;  it  must  not  contain  floculi ; 
it  should  not  precipitate  on  standing;  it  should,  in  short,  be  a 
stable  hydrosol.  Since  there  seem  to  be  some  differences  in  the 
fermentability,  i.e.,  in  the  chemical  resistance  of  starches  from 
different  plants,  all  the  material  in  a  series  of  experiments  should 
be  prepared  from  one  source,  and  preferably  at  one  operation. 
For  studies  of  amylase  of  vegetable  origin,  glycogen  is  sometimes 
preferable  to  starch,  in  that  it  is  very  soluble  in  water  and  seems 
to  present  fewer  sub-stages  in  the  process  of  cleavage.  In  the 
use  of  glycogen,  however,  the  absence  of  maltase  from  the  amy- 
lase under  investigation  must  be  determined.  For  investigations 
with  mammalian  amylase  glycogen  cannot  be  used,  because  all 
animal  extracts  and  secretions  containing  amylase  contain  also 
maltase,  and  the  fermentation  of  the  maltose  could  not  be  meas- 
ured. Against  the  availability  of  glycogen  under  any  circum- 
stances are  the  difficulties  of  preparation  in  quantity,  and  its 
tendency  to  denaturation  during  the  manipulations  attending  its 
isolation  and  purification. 

The  Mett  method,  the  use  of  capillary  tubes  filled  with  starch 
gel,  is  entirely  unadapted  to  studies  on  the  cleavage  of  starch, 
since  only  the  liquefaction  of  starch  is  measured  with  this 
method.  Even  this  cannot  in  my  experience  be  measured  with 
regular  and  satisfactory  results. 


156  University  of  California  Publications.      [PATHOLOGY 

The  measurement  of  the  progress  of  the  hydrolysis  of  starch 
is  an  unsatisfactory  operation.  The  use  of  color  reactions,  as 
those  with  iodine,  is  wholly  unadapted  to  accurate  work ;  one 
cannot  in  this  manner  even  determine  precisely  the  moment  of 
disappearance  of  the  starch,  and  the  successive  stages  are  still 
less  satisfactorily  demarcated.  The  only  feasible  measurement  is 
that  of  the  maltose.  This  cannot  be  determined  by  polariscopy, 
though  the  reasons  for  this  have  not  been  clearly  worked  out. 
The  maltose  must  be  determined  by  chemical  methods,  best  by 
reduction  of  copper  in  Fehlings  solution.  It  must  at  the  outset 
be  realized  that  the  technique  of  the  estimation  of  maltose  by 
Fehling's  solution  has  not  been  developed  as  in  the  case  of  glu- 
cose, which,  thanks  to  Pflueger^  can  now  be  estimated  with  great 
accuracy.  A  preliminary  estimation  must  be  always  made  to 
determine  whether  the  substrate  solution  possess  any  reducing 
power,  since  it  is  a  fundamental  assumption  in  the  equation  that 
when  t  =  0,  x  is  likewise  0.  In  the  calculation  of  the  findings  the 
equation  must  be,  so  to  speak,  reversed,  as  suggested  by  Henri : 
A  stands  for  the  total  maltose  at  the  end  of  the  reaction,  x  for 
the  maltose  formed  in  the  time  t.  That  this  use  of  the  equation 
is  not  theoretically  entirely  justified  is  seen  in  the  fact  that  the 
reaction  is  not  complete,  but  the  uncompleted  portion  is  present 
in  the  system  in  the  form  of  a  sub-stage  product,  a  dextrine,  and 
not  in  the  form  of  the  substrate  starch.  Nevertheless  the  results 
seem  fairly  satisfactory,  in  that  they  conform  to  the  theoretical 
expectations. 

Kinetics  of  the  Fermentation.  The  progression  of  the  reac- 
tion in  the  conversion  of  starch  into  sugar  follows  in  general 
terms  the  regular  law.  Brown  and  Glendenning,1  employing  an 
amylase  from  malt,  obtained  figures  that  were  a  little  too  rapid 
to  be  accurately  represented  in  a  logarythmal  curve,  but  which 
gave  fairly  good  concordance  for  the  constants  when  calculated 
with  the  empirical  equation  of  Henri :  C  --  =  1  log  ^~- .  Henri,1 
on  the  contrary,  working  with  ferments  from  malt  and  from  the 
pancreas,  obtained  results  closely  in  accord  with  the  logarythmal 
curve.  In  both  instances  these  results  were  obtained  only  with 
dilute  solutions ;  but  for  the  ferment  from  the  malt  the  conclu- 
sions were  valid  only  for  solutions  below  0.75  per  cent.,  while 


VOL.  1]  Taylor — On  Fermentation.  157 

with  the  pancreatic  ferment  solutions  could  be  employed  up  to 
2  per  cent.  The  acceleration  bore  a  relation  proportional  to  the 
quantity  of  ferment. 

I  have  tested  the  diastatic  ferment  of  saliva,  using  the  fresh 
human  secretion.  Comparative  researches  are  difficult  because 
constancy  in  the  ferment  cannot  be  maintained  through  a  pro- 
longed secretion.  The  substrates  were  one-quarter,  one-half,  and 
three-quarter  per  cent,  solutions  of  soluble  starch,  mixed  with 
one-tenth  vol.  of  fresh  saliva  obtained  by  the  mastication  of  pa- 
raffine.  The  saliva  was  distinctly  alkaline;  no  other  alkali  was 
added. 

Fairly  satisfactory  experimentation  may  be  done  with  the 
salivary  amylase  if  a  few  precautions  are  followed.  The  saliva 
must  be  obtained  from  glands  that  have  had  several  hours'  rest. 
The  mouth  should  be  several  times  washed  with  an  antiseptic 
solution,  as  salicylic  acid  or  benzoyl-acetyl-peroxide.  The  secre- 
tion of  saliva  should  be  stimulated  by  the  mastication  of  pure 
paraffine.  The  first  portions  of  the  saliva  should  be  rejected; 
the  mucus  content  is  high,  the  bacterial  count  high,  the  ferment 
concentration  low.  The  filtered  saliva  should  be  added  to  the 
starch  solution  at  about  30-35°.  At  this  temperature  saccharifi- 
cation  is  less  rapid  than  at  45°  ;  inactivation  of  the  ferment  is 
also  less  active.  For  tests  of  moderate  length,  the  addition  of  an 
antiseptic  is  not  necessary.  At  stated  intervals  portions  are  re- 
moved, rapidly  heated  to  boiling,  and  then  added  to  Fehling's 
solution  in  a  water  bath  (according  to  Pflueger),  and  the  reduc- 
tion of  copper  measured  gravimetrically.  It  is  not  advisable  to 
carry  the  experiment  beyond  the  saccharification  of  about  half 
the  substrate,  since  the  maltase  (always  present)  will  more  and 
more  appreciably  invert  the  disaccharide  into  glucose.  It  is  a 
curious  fact  that  the  maltase  of  saliva  will,  convert  maltose  into 
glucose  during  the  course  of  such  an  experiment  much  more  rap- 
idly in  the  case  of  glycogen  than  in  the  case  of  starch.  For  this 
reason  the  results  with  starch  are  quite  satisfactory,  those  with 
glycogen  are  quite  irregular.  The  active  concentration  of  amy- 
lase in  saliva  is  so  different  in  different  times  of  secretion  that 
comparisons  cannot  be  made  except  between  tests  done  with  the 


158  University'  of  California  Publications.      [PATHOLOGY 

same  saliva  at  different  concentrations  of  substrate  or  ferment. 
The  results  of  one  of  such  series  were  as  follows  : 

Substrate  i  %  .     Temperature  35? 


t  (min.) 

30 

45 

60 

75 

90 

120 

150 

180 

C  (x  10~6) 

490 

465 

455 

470 

465 

455 

560 

455 

Substrate  i  %  . 

t  (min.) 

30 

45 

60 

75 

90 

120 

150 

180 

C  (x  10'6) 

430 

420 

390 

415 

405 

395 

430 

410 

Substrate  i  %  . 

t  (min.) 

30 

45 

60 

75 

90 

120 

150 

180 

C  (x  10~6) 

390 

370 

385 

390 

480 

370 

365 

370 

The  constants  were  calculated  according  to  the  equation  C  = 

-  log  A       used  in  the  reversed  manner  suggested  by  Henri:     A 

t  A—'X 

is  the  total  product  when  the  reaction  is  complete,  and  x  the 
amount  of  product  formed  in  the  time  t. 

The  figures  cannot  be  compared  with  Henri  's2  figures  for  pan- 
creatic juice  because  there  is  no  way  to  allow  for  the  differences 
in  the  conditions  of  experimentation.  His  constants  for  a  0.75 
per  cent,  solution  at  18°  were: 

t  (min.)  34  55  94  133  273 
C  (xlO-~»)  238  .  222  227  212  200 

The  constants  are  quite  concordant  in  each  series,  but  they 
are  not  in  agreement  in  the  several  series.  We  here  encounter  a 
concrete  illustration  of  an  experimental  finding  that  is  frequently 
obtained  in  quantitative  studies  in  fermentations,  and  which  has 
not  been  sufficiently  appreciated.  While  we  may  obtain  in  the  • 
calculations  of  a  series  of  measurements  in  a  particular  experi- 
ment constants  that  exhibit  a  satisfactory  uniformity,  this  uni- 
formity is  not  to  be  obtained  in  several  series  carried  out  with 
the  same  ferment  concentration  but  with  a  different  substrate 
concentration.  Theoretically,  all  concentrations  of  the  substrate 
(within  certain  limits)  should  yield  with  the  same  concentration 

•  >i'  ferment  uniform  figures  for  the  velocity  constants.     The  fig- 
ures given  above  for  amylase  illustrate  the  failure  to  do  this. 
For  this  failure  to  realize  the  theory  many  possible  explanations 

/?  Wt/  may  be  suggested,  but  of  these,  three  only  are  of  fundamental 
import.  It  is  in  the  first  place  possible  that  the  intensity  of  the 
ejizyme  is  a  function  not  only  of  its  own  concentration,  but  also 


Taylor — On  Fermentation.  159 

of  the  substrate  concentration.  To  this  factor  of  intensity  of 
ferment  action  Visser  has  recently  given  special  attention,  but 
I  do  not  think  he  has  been  able  to  furnish  an  actual  elucidation 
of  the  relationship.  That  the  non-uniformity  in  the  constants  in 
different  series  of  substrate  concentrations  cannot  be  reduced  to 
a  variation  in  the  intensity  of  the  ferment  action  is  made  highly 
probable  by  the  fact  that  the  acceleration  of  the  reaction  in  dif- 
ferent series  with  the  same  substrate  concentration  is  propor- 
tional to  the  concentration  of  ferment.  A  concrete  illustration 
will  make  this  clear.  In  three  series  of  tests  with  respectively 
one-quarter,  one-half,  and  three-quarter  per  cent,  solutions  of 
soluble  starch,  and  with  a  uniform  ferment  concentration,  the 
constants  will  be  fairly  uniform  in  each  series,  but  not  at  all 
uniform  in  the  several  series.  For  each  of  these  substrate  con- 
centrations, on  the  other  hand,  doubling  the  ferment  will  double 
the  acceleration.  These  facts  are  not  in  harmony  with  the  hy- 
pothesis that  the  lack  of  uniformity  in  the  constants  in  series  of 
only  slightly  different  substrate  concentration  is  due  to  varia- 
tions in  the  intensity  of  ferment  action. 

The  second  explanation  would  rest  the  variation  in  constants 
on  the  factor  of  the  coefficient  of  distribution  in  the  relations  of 
ferment  and  substrate  in  the  system.  That  variations  in  the  con- 
centration of  the  substrate  might  lead  directly  to  variations  in 
this  coefficient,  and  also  in  the  velocity  of  the  distribution,  is  ap- 
parent. That  variations  in  the  mass  of  ferment  would  likewise 
lead  to  alterations  in  the  coefficient  is  of  course  true,  but  the 
mass  of  ferment  in  an  experiment  is  so  small  in  contrast  to  the 
mass  of  the  substrate  that  we  would  not  expect  any  deviations 
due  thereto  to  be  apparent  in  the  measurements.  At  first  sight, 
it  might  be  expected  that  this  factor  should  be  tested,  on  the  as- 
sumption that  variations  in  the  constants  ought  to  be  propor- 
tional to  variations  in  the  substrate  concentration.  This  could 
not,  however,  be  expected,  unless  the  coefficient  of  distribution 
and  the  velocity  of  distribution  were  functions  of  the  viscosity, 
which  they  are  not ;  they  are  in  large  part  functions  of  the  chem- 
ical qualities  of  the  reacting  bodies,  and  these  qualities  for  the 
substances  under  consideration  cannot  be  determined.  While 
therefore  the  extent  of  this  factor  cannot  be  even  guessed  at,  it  is 


160  University  of  California  Publications.      [PATHOLOGY 

certain  that  variations  in  the  substrate  concentration  might  be 
expected  to  lead  to  variations  in  the  velocity  constants  as  the  re- 
sult of  the  operation  of  the  factors  o  fthe  coefficient  and  velocity 
of  distribution. 

The  third  reason  for  non-uniformity  in  the  constants  lies  in 
the  fact  that  we  are  dealing  with  a  reaction  in  many  stages.  On 
the  assumption  that  the  enzymic  acceleration  consists  of  a  series 
of  intermediary  reactions  in  each  stage  (and  we  are  able  to  meas- 
ure only  the  last  stage),  a  uniformity  of  progression  could  not 
be  expected  with  a  fixed  concentration  of  catalysor  in  series  of 
different  concentrations  of  substrate.  Experience  with  reactions 
in  many  stages  has  in  many  lines  of  chemical  investigation  led 
to  uninterpretable  results,  so  that  we  do  not  here  face  a  difficulty 
peculiar  to  fermentations.  That  this  factor  may  be  of  determin- 
ing importance  is  suggested  by  the  fact  that  a  similar  lack  of 
uniformity  in  the  constants  for  different  substrate  concentra- 
tions is  observed  in  the  hydrolysis  of  starch  with  acids. 

From  the  figures  it  appears  that  the  constants  diminish  as 
the  substrate  concentration  increases.  The  averages  for  the  three 
series  are:  one-quarter  per  cent.,  494;  one-half  per  cent.,  415; 
and  three-quarters  per  cent.,  390.  Between  the  reductions  in  the 
constants  and  the  increases  in  the  substrate  concentrations,  how- 
ever, no  mathematical  relations  exist. 

In  the  above  considerations,  it  has  been  assumed  that  the  fer- 
ment was  not  inactivated  or  destroyed  in  the  experiment.  This 
condition  may  be  approximately  attained  with  amylase.  If  the 
ferment,  however,  were  less  stable,  further  variations  in  the  con- 
stants would  be  expected,  since  in  general  terms  the  hydrolysis 
of  the  ferment  (its  destruction)  is  proportional  to  the  active 


t/AJL, 


mass  °f  tne  ferment,  and  this  is  in  general  terms  inversely  pro- 


ortional  to  the  untransf ormed  substrate ;  the  inactivation  of  the 
ferment  would  therefore  proceed  differently  with  different  con- 
;entrations  of  substrate. 

-    In  all  the  work  on  starch  it  has  been  found  that  with  solu- 
//  'A/ tions  of  high  concentration  the  transformation  in  the  unit  of 

time  did  not  correspond  to  the  mass  of  substrate ;  it  was,  in  fact, 
usually  constant  in  time.  This  fact  has  been  used  against  the 
idea  that  the  fermentation  of  these  bodies  bore  a  close  resem- 


VOL.  1]  Taylor — On  Fermentation.  ,Jb         161 

blance  to  the  acid  hydrolysis  of  these  bodies.  As  a  matter  of 
fact,  the  results  do  not  support  the  objection  at  all.  All  along 
the  line  of  work  in  catalysis  it  has  been  recognized  that  high  con- 
centrations of  the  substrate  disturbed  the  relations.  In  some  of 
the  published  work  the  concentrations  have  been  enormous: 
Duclaux  used  solutions  from  10  to  40  per  cent.,  Brown  from  5  to 
20  per  cent. !  The  higher  concentrations  given  amount  to  veri- 
table pastes.  Now  one  must  distinguish  between  the  total  mass 
of  the  substrate  and  the  active  mass.  One  cannot  resist  the  com- 
parison with  the  active  mass  and  the  total  concentration  of  an 
acid.  The  acid  acting  as  a  catalysor  is  active  only  in,  proportion 
to  its  dissociation ;  and  certainly  there  is  some  such  distinction 
in  organic  molecules,  although  we  may  possess  as  yet  no  measur- 
able notion  of  the  physical  or  chemical  state  to  which  this  activity 
belongs.  It  must  not  be  imagined  that  the  law  of  Wilhelmy  holds 
for  the  highest  concentrations  of  sugar,  not  at  all.  If  one  were 
titrating  a  concentrated  solution  of  sulphuric  acid  with  barium 
hydrate,  one  could  add  the  alkali  without  much  reduction  in  the 
conductivity  until  a  dilution  to  less  than  15  per  cent,  had  been 
reached;  as  each  successive  ion  of  S04  was  combined  with  the 
barium  and  passed  out  of  solution,  another  molecule  of  the  acid 
would  be  at  once  dissociated,  so  that  the  active  concentration 
would  diminish  but  very  slowly  for  a  long  time.  And  in  an  analo- 
gous manner  we  must  consider  that  if  a  ferment  were  acting  upon 
a  concentrated  solution  of  starch,  as  each  active  molecule  became 
hydrolyzed  its  place  would  be  taken  by  another  molecule  from 
the  inactive  excess,  and  thus  the  active  mass  would  remain  con- 
stant and  the  amount  of  inversion  in. the  unit. of  time  would .xe- 
main  constant.  But  in  dilute  solutions,  as  has  been  uniformly 
noted,  the  transformation  is  proportional  to  the  mass — simply 
because  under  these  conditions  all  the  substrate  is  active  in  the 
reaction  sense.  Philoche2  has  recently  reported  upon  a  study  of 
the  acceleration  of  the  saccharifieation  of  soluble  starch  by  vege- 
table amylase,  derived  from  malt  and  from  the  taka  yeast.  Em- 
ploying solutions  of  2  per  cent,  substrate,  she  found  that  for  a 
time  the  constants  rapidly  descended,  then  became  uniform  and 
remained  so  during  the  experiment.  It  is  possible  that  had  the 
initial  concentration  of  the  substrate  been  lower,  the  constants 


162  University  of  California  Publications.      [PATHOLOGY 

would  have  been  uniform  during  the  entire  experiment,  though 
the  author  states  that  the  initial  drop  was  observed  in  many  series 
of  digestions. 

In  a  calculation  of  the  relation  of  the  mass  to  the  reaction 
velocity  in  any  reacting  system,  the  total  mass  must  be  known  to. 
be  identical  wit.Ti  thp  apf.jyp  mass,  This  cannot  be  demonstrated 
for  solutions  of  substances  like  starch  and  protein.  For  these 
stable  or  pasedo-colloids,  we  may  in  all  probability  assume  that 
one  of  two  conditions  of  state  exist :  (a)  The  substance  is  capable 
of  a  slight  degree  of  true  solution  (just  as  a  fat  is)  ;  the  larger 
part  of  the  substance,  however,  is  present  suspended  as  a  colloid. 
Under  this  interpretation,  we  should  assume  that  the  reaction  is 
a  function  largely  of  the  fraction  in  true  solution,  though  the 
velocity  would  not  depend  upon  it  alone  since  the  reaction  occurs 
in  the  film  of  contact  of  the  two  phases,  the  watery  and  the  col- 
loidal phase.  (6)  The  substance  is  incapable  of  any  appreciable 
true  solution.  Under  this  interpretation  there  would  be  a  reaction 
of  equilibrium  and  partition  between  the  colloid,  the  water  and 
the  catalysor.  The  present  evidence  tends  to  indicate  that  the 
laws  of  mass  action,  equilibrium,  and  partition  tend  to  hold  for 
the  stable  colloMs,  like  starch  and  protein,  but  do  not  tend  to  hold 
for  the  typical  unstable  colloids,  like  the  metallic  hydrosols.  Un- 
der either  point  of  view,  variations  in  the  substrate  concentrations 
would  be  expected  to  lead  to  variations  in  the  intensity  of  the 
reaction ;  in  the  case  of  a  suspended  colloid  because  it  would  pro- 
duce an  alteration  in  the  relation  of  equilibrium ;  in  the  case  of 
a  true  solution  because  it  would  bring  about  an  alteration  in  the 
active  mass.  That  reactions  may  display  great  variations  as  the 
result  of  variations  in  concentrations,  even  in  systems  of  simple 
inorganic  reaction,  is  well  illustrated  in  the  recent  observation  of 
Sheppard.  When  hydroxylamine  and  silver  oxide  react  in  a 
system  at  moderate  concentration,  the  reaction  follows  the  equa- 
tion —  2  NH2OH  -f  Ag20  =  2  Ag  +  N2  +  3  H20;  when  the  sys- 
tem is  in  high  dilution,  howrever,  the  reaction  follows  the  equation 
-  2  NH2OH  -f  2  Ag20  =  4Ag  +  N20  -f  3  //,. 

Since  the  velocity  of  the  reaction  of  the  fermentation  of 
starch   follows   the   common    law    C=~r log  .    -  it  is  apparent 

I  j?L'      S/ 

that  viewed  from  the  point  of  view  of  the  theoretical  equation  of 


VCL.  1]  Taylor — On  Fermentation.  163 

Henri  to  be  described  under  invertase,  the  coefficients  m  and  n 
must  be  equal,  and  thus  the  progress  of  the  reaction  is  not  devi- 
ated from  the  logarythmal  curve. 

Digestion  experiments  with  amylase  exhibit  well  the  so-called 
false  equilibrium  of  fermentations.  After  the  transformation 
has  come  to  a  standstill,  the  reaction  may  be  reinaugurated  by 
the  addition  of  more  of  the  substrate  or  by  the  removal  of  the 
products  (Morris  &  Glendenning) .  On  account  of  the  resistance 
of  amylase  to  hydrolysis,  these  experiments  can  usually  be  re- 
peated a  number  of  times  in  one  system.  That  the  products  de- 
press the  reaction  through  the  relations  of  the  mass  law  rather 
than  by  any  chemical  action  upon  the  ferment  may  be  shown  by 
the  following  experiment.  Two  systems  are  prepared,  one  in  a 
simple  container,  the  other  in  a  dialyser.  The  reaction  in  the 
dialyser  will  be  more  rapid  and  more  fully  completed  than  in 
the  simple  experiment.  If  now  after  the  completion  of  the  re- 
actions in  both,  the  sugar  be  removed  from  the  simple  test  by 
dialysis,  and  both  residues  then  tested  for  strength  of  ferment,  it 
will  be  found  that  both  have  the  same  ferment  content. 

Relation  of  ferment  concentration  on  acceleration  of  reaction. 
This  has  usually  been  stated  by  Pawlow  to  be  proportional  to 
the  square  root  of  the  ferment,  the  results  having  been  obtained 
with  the  Mett  method.  Henri3  has  found  the  transformation  by 
vegetable  amylase  to  be  proportional  to  the  ferment  concentra- 
tion, and  I  .have  obtained  the  same  result  with  salivary  amylase. 
Thus : 

Substrate  concentration  one-quarter  per  cent,  a  has  1/10  vol. 
saliva ;  b  has  1/5  vol.  saliva.  The  times  are  the  periods  necessary 
to  complete  the  given  fractions  of  the  total  conversion. 

i  4  f 

a  (row.)  32  75  120 

ft  18  40  70 

Brown  and  Glendenning,2  however,  found  in  their  experi- 
ments with  salivary  amylase  that  the  acceleration  was  propor- 
tional to  the  square  root  of  the  ferment  concentration. 

Influence  of  temperature.  This  is  strikingly  different  for  the 
processes  of  liquefaction  and  saccharification.  The  optimum  tem- 
perature for  the  process  of  liquefaction  lies  high,  between  60-70°, 


164  University  of  California  Publications.      [PATHOLOGY 

for  some  preparations  over  75°.  At  these  temperatures,  however, 
the  ferment  is  rapidly  destroyed,  so  that  the  time  gained  in 
liquefaction  is  more  than  lost  in  saccharification,  and  in  practice, 
when  such  a  high  temperature  is  employed  for  liquefaction,  a  new 
quantum  of  ferment  is  added  for  the  hydrolysis.  The  velocity 
for  the  hydrolytic  acceleration  rises  in  general  conformity  to 
the  law  for  the  increase  of  reaction  velocity  with  temperature 
until  40°,  from  which  point  until  60°  the  reaction  is  very  rapid, 
only  to  fall  rapidly  beyond  60°.  Different  preparations  of  amy- 
lase,  however,  vary  widely  in  these  regards.  For  the  animal 
amylases  the  optimum  temperature  is  some  15°  to  20°  lower  for 
the  reaction  of  hydrolysis,  while,  for  salivary  diastase  at  least, 
there  is  no  marked  difference  in  the  optimum  temperatures  for 
the  two  processes.  Solutions  of  amylase  bear  heating  badly  above 
70° ;  dessicated  preparations  may  be  heated  to  over  100°  for  a 
long  time  without  injury,  though  here  again  different  prepara- 
tions vary. 

Reversion  of  reaction.  The  hydrolytic  cleavage  of  starch, 
whether  accelerated  by  acids  or  ferments,  or  whether  accom- 
plished by  heat  alone,  is  practically  never  completed.  Starch 
does  not  remain  in  the  system,  but  a  certain  proportion  of  dex- 
trine remains.  The  amount  of  dextrine  remaining  depends  to 
some  extent  on  the  concentration  of  the  substrate;  at  high  con- 
centrations much  more  remains  than  at  high  dilutions.  In  a 
one-quarter  per  cent,  solution  not  over  1  to  5  per  cent,  remains ; 
at  high  concentrations  as  much  as  one-fifth  may  remain.  Com- 
plete reactions  have  been  reported  as  accomplished  by  the  addi- 
tion of  fresh  portions  of  ferment,  but  I  have  personally  never 
been  able  to  recover  the  full  quota  of  maltose.  The  matter  can- 
not be  decided  on  the  basis  of  a  direct  calculation  of  the  amount 
of  maltose  to  be  derived  from  a  known  quantity  of  starch,  since 
it  is  not  known  exactly  how  much  maltose  a  unit  of  starch  will 
yield,  though  the  ratio  is  about  10 : 9.  The  demonstration  of  the 
incompleteness  of  the  reaction  is  to  be  made  by  the  demonstration 
of  an  increase  in  the  maltose  after  the  larger  mass  of  the  product 
has  been  removed  from  the  system  by  dyalysis ;  if  the  ferment 
be  not  inactivated,  this  experiment  succeeds.  Since  it  is  an  ob- 
vious advantage  in  the  mathematical  interpretation  of  the  results 


VOL.  l]  Taylor — On  Fermentation.  165 

to  have  the  reaction  as  nearly  complete  as  possible  (i.e.,  to  have 
the  station  of  equilibrium  in  starch  =  maltose  as  far  to  the  right 
as  possible,  and  the  disturbing  influence  of  the  reversed  reaction 
as  small  as  possible),  we  have  an  additional  reason  for  the  em- 
ployment of  high  dilutions  of  the  substrate. 

A  clear  reversion  of  the  saccharin" cation  or  of  the  liquefaction 
of  starch  has  not  been  described.  There  exist,  however,  some 
studies  by  Wolff,  Fernbach,  and  Maquenne  that  point  strongly 
in  this  direction.  They  have  constated  after  the  action  of  dias- 
tase the  reformation  of  coagulable  starch  and  of  a  body  giving 
the  iodine  stain.  The  authors  themselves  seem  to  incline  to  the 
view  that  the  reversion  may  be  the  work  of  a  totally  different 
ferment.  The  matter  is  still  under  investigation,  and  should  it 
transpire  that  true  reversion  has  occurred,  we  may  confidently 
believe  the  reversion  to  have  been  caused  by  the  same  ferment 
rather  than  by  a  different  ferment.  The  formation  of  glycogen 
by  reversion  of  ferment  activity  has  been  described  by  Cremer. 
He  allowed  a  yeast  amylase,  free  of  glycogen,  to  act  upon  a  con- 
centrated solution  of  glucose.  A  polysaccharide  was  formed  that 
gave  the  qualitative  reactions  of  glycogen.  It  may  not  have  been 
identical  with  glycogen,  but  it  was  a  starch-like  body,  and  with 
that  the  synthesis  of  a  polysaccharide  by  acceleration  of  rever- 
sion through  ferment  action  was  accomplished.  We  may  in  all 
probability  regard  the  formation  of  glycogen  from  sugar  as  an 
instance  of  the  formation  of  a  poly-saccharide  from  a  mono- 
saccharide  by  enzymic  acceleration  of  the  reversed  reaction. 
That  acid  reversion  of  the  hydrolysis  exists  has  been  made  very 
probable  by  Wohl,  who  has  further  shown  that  under  conditions 
of  high  concentration  of  the  substrate  the  acid  hydrolysis  is  al- 
ways a  limited  reaction. 

Conditions  of  activity.  Amylases  of  vegetable  origin  seem 
to  operate  more  rapidly  at  a  slightly  acid  reaction,  though  fer- 
mentation occurs  actively  at  a  neutral  reaction.  The  animal 
amylases,  on  the  contrary,  operate  in  a  faintly  alkaline  or  neutral 
reaction  better  than  in  an  acid  reaction.  According  to  Foa,  the 
alkalinity  of  the  saliva  is  about  X/80000.  For  both  classes  a 
slight  excess  of  the  alkali  or  acid  is  harmful,  and  the  optimum 
concentration  seems  to  be  rather  narrow.  The  influence  of  the 


166  University  of  California  Publications.      [PATHOLOGY 

reaction,  however,  varies  notably  with  the  temperature,  as  well 
as  with  the  concentrations  of  the  substrate  and  the  ferment.  Of 
quantitative  data  there  are  none  of  value.  In  all  the  published 
work  on  the  degree  of  acidity  and  alkalinity  employed  in  the  ex- 
periments with  amylase,  that  degree  of  reaction  has  been  deter- 
mined either  by  adding  a  measured  quantity  or  by  an  end  titra- 
tion.  The  procedures  of  course  do  not  give  information  upon 
the  point  desired,  the  concentration  of  hydrogen  or  hydroxylion 
ions.  The  action  of  acids  and  alkalies  in  the  concentrations  em- 
ployed is  in  the  role  of  a  zymoexciter,  not  that  of  a  second  cata- 
lysor  whose  acceleration  is  added  to  that  of  the  ferment.  Under 
the  circumstances  we  do  not  know  whether  it  may  not  be  in  part 
the  undissociated  molecule  that  acts  as  the  zymoexcitor;  just  as 
many  other  undissociated  substances  may  thus  act.  The  entire 
situation  is  unclear ;  but  it  is  certain  that  none  of  the  experiments 
ever  determined  exactly  what  active  acidity  or  alkalinity  was 
being  used.  This  accelerating  action  of  acid  for  vegetable  dias- 
tase seems  to  be  confined  to  the  saccharification,  and  does  not 
affect  the  process  of  liquefaction. 

Many  other  substances  depress  the  activity :  salts  of  lead, 
mercury,  barium,  arsenic,  iron,  alcohols,  phenols,  formaldehyde. 
Chloroform  has  little  influence;  toluol  has  none.  Stimulating, 
on  the  other  hand,  are  the  phosphates  of  calcium  and  ammonium, 
the  common  alumns,  aluminum  acetate,  and  asparagin.  With  the 
exception  of  the  asparagin,  these  substances  act  largely  upon  the 
process  of  liquefaction.  Amido  acids  as  a  group  stimulate  the 
action  of  amylase,  while  acid  amides  depress  effront.  There  are 
well  defined  optimum  concentrations  for  these  substances.  For 
both  the  stimulating  and  the  depressing  substances  the  action  is 
in  a  general  way  inversely  to  the  mass  of  ferment  present.  There 
is  an  enormous  literature  upon  the  subjects  of  the  action  of  va- 
rious substances  on  amylase.  Though  different  experimenters 
have  carried  out  elaborate  series  of  experiments,  their  results 
have  been  often  very  contradictory,  both  in  the  quantitative  and 
in  qualitative  details.  The  reason  for  this  lies  in  the  fact  that 
the  reactions  of  a  ferment  to  different  substances  and  the  reac- 
tions of  various  substances  upon  a  ferment  are  to  a  marked  de- 
gree different  with  different  preparations  of  the  ferment,  just  as 


VOL.  l]  Taylor — On  Fermentation.  167 

are  its  resistances  to  heat,  to  hydrolysis,  etc.,  relations  that  are 
well  known  to  hold  for  all  colloids,  for  the  metallic  colloids  as 
well  as  for  ferments.  Under  these  circumstances,  investigations 
on  the  actions  of  different  chemical  substances  on  amylase  pos- 
sess an  approximate  value  only. 

Amylase  is  more  rapidly  destroyed  by  being  heated  in  simple 
solution  than  when  heated  in  the  presence  of  starch.  Under  fa- 
vorable conditions  of  temperature,  concentration,  and  reaction, 
amylase  is  destroyed  to  but  a  very  slight  extent  during  the  course 
of  a  fermentation ;  it  is  so  resistant  to  hydrolysis  that  it  may  be 
said  to  display  for  practical  purposes  the  integrity  characteristic 
of  a  true  catalysor,  that  it  shall  not  be  altered  by  the  reaction 
it  accelerates.  In  pure  solutions  the  ferment  is  destroyed  with 
moderate  rapidity ;  when  dessicated  it  may  be  kept  indefinitely, 
and  is  then  resistant  to  high  temperatures.  The  best  method  of 
preserving  amylase  is  to  keep  it  mixed  with  starch  at  a  low  tem- 
perature; under  such  conditions  one  can  prepare  enough  for  a 
series  of  experiments,  and  very  little  change  will  occur. 

Chemical  properties.  The  chemical  attributes  of  amylase  are 
not  well  known.  There  has  been  apparently  only  one  prepara- 
tion of  a  reasonably  pure  amylase  of  vegetable  origin,  by  Wro- 
blowsky,  and  none  of  animal  amylase.  The  substance  isolated 
by  Wroblowsky  was  colorless,  very  soluble  in  water,  of  neutral  re- 
action, not  coagulable  by  heat,  was  precipitated  by  a  50%  ammo- 
nium sulphate  and  by  the  salts  of  lead  and  mercury,  responded 
to  the  biuret,  Millon  and  Xanthoproteic  reactions,  and  was  pre- 
cipitated by  tannic  acid  and  alcohol  without  denaturation.  It  is 
not  crystalline,  very  slightly  diffusible  if  at  all  in  the  practical 
sense,  but  may  be  filtered  (though  with  loss)  through  infusorial 
filters.  It  clings  to  colloidal  structures,  and  cannot  be  filtered 
through  paper  without  some  loss.  It  contains  some  16.5  per  cent, 
of  nitrogen,  and  is  digestible  by  pepsin,  less  readily  by  trypsin. 
The  products  of  the  digestion  are  amido  acids,  leucine,  tyrosin 
and  arginine  having  been  isolated.  In  general  terms  it  may  be 
named  an  albumose. 


168  University  of  California  Publications.      [PATHOLOGY 


INVERTASE  AND  LACTASE. 

The  inversion  ferment  for  cane  sugar  was  discovered  in  beer 
yeast  by  Doebereiner  and  Mitscherlich ;  it  was  first  isolated  by 
Berthelot.  It  is  now  known  to  occur  almost  universally.  In  the 
higher  animals  it  is  present  in  the  saliva,  in  the  secretions  of  the 
small  intestines,  and  in  the  liver.  It  seemingly  displays  a  less 
wide  distribution  in  the  animal  kingdom  than  amylase,  probably 
because  it  has  been  less  often  sought  for.  Cohnheim  found  it 
present  in  all  echinoderms  examined.  It  has  been  found  in  the 
eggs  of  crustaceans.  In  the  vegetable  world  it  is  probably  the 
most  widely  distributed  of  ferments.  Unlike  the  amylase,  it  is 
found  to  a  less  extent  in  the  seeds  and  to  a  greater  extent  in  the 
growing  parts,  buds,  and  leaves.  Kastle  and  Clark  in  a  recent 
study  examined  nineteen  species  belonging  to  fourteen  families 
and  found  invertase  in  all.  It  is  often  found  in  tissues  that 
possess  no  sacchrose.  It  is  found  frequently  in  bacteria,  not  only 
in  the  yeasts  but  also  in  the  bacilli  and  cocci.  The  bacteria  that 
have  the  function  of  lactic  acid  fermentation  seem  to  be  able  to 
accelerate  also  the  inversion  of  sacchrose.  Among  the  disease- 
producing  micro-organisms  the  following  possess  in  a  prominent 
manner  the  faculty  of  inverting  cane  sugar  and  then  fermenting 
the  hexose:  Pneumo-bacillus  of  Friedlaender,  Bac.  diphtheric, 
Bac.  malignant  oedema,  the  colon  bacillus,  and  the  proteus  vul- 
garis.  Doubtless  there  are  many  others.  The  named  micro- 
organisms are  also  able  to  invert  milk  sugar,  but  have  no  power 
to  accelerate  the  inversion  of  maltose. 

Whether  these  various  invertases  are  identical  is  not  known. 
Of  all  ferments,  invertases  from  different  sources  display  the 
most  notable  variations  in  physical  and  chemical  qualities.  Dif- 
ferent invertases  display  variations  of  as  much  as  40°  in  their 
optimum  temperatures ;  some  are  very  sensitive  to  acids  and  heat, 
others  quite  resistant;  some  are  dextro-rotary,  others  do  not  po- 
larize light;  some  solutions  are  very  colloidal,  others  not  at  all 
so;  some  preparations  are  diffusible,  others  scarcely  filterable. 
Very  interesting  are  the  differences  in  the  optimum  temperatures 
of  the  two  common  yeasts  of  beer;  the  invertase  of  surface-fer- 
mentation yeast  has  an  optimum  temperature  of  some  20-30° 


VOL.  l]  Taylor — On  Fermentation.  169 

higher  than  that  of  sediment-fermentation  yeast;  the  one  is  de- 
rived from  a  yeast  with  strong  predeliction  for  oxygen,  the 
other  from  a  yeast  with  a  slight  predeliction  for  oxygen,  and 
nevertheless  these  yeasts  represent  cultural  deviations  from  one 
parent  stock.  Invertase  from  one  source  also  will  display  marked 
variations  in  properties,  depending  upon  differences  in  the  his- 
tory of  the  culture  and  the  methods  of  preparation.  These  facts 
indicate  the  futility  of  present  speculations  on  the  unity  or  multi- 
plicity of  invertases.  It  is  known  that  invertase  is  able  to  accel- 
erate the  inversion  of  sacchrose  alone,  not  of  lactose  or  maltose. 
Mnltase  is  apparently  of  less  wi^e  distribution,  though  it  is  en- 
countered largely  in  grains,  yeasts,  and  in  the  growing  parts  of 
plants.  It  was  discovered  by  Gusenier,  though  the  cleavage  of 
maltose  in  urine  had  been  described  by  Bechamp.  In  mamma- 
lians it  has  been  shown  to  exist  in  the  saliva,  pancreatic  juice, 
succus  entericus,  liver,  blood,  and  urine.  The  current  statements 
that  maltase  is  able  to  act  upon  starch  and  glycogen  is  surely  an 
error  based  upon  an  admixture  of  amylase.  Lactase,  the  ferment 
of  milk  sugar,  was  first  described  by  Beyerinck,  who  found  it  in 
the  kephyr  yeast.  It  is  now  known  to  be  present  in  many  yeasts. 
It  has  not  yet  been  directly  shown  to  be  present  in  animals  apart 
from  the  intestinal  secretion,  but  the  mere  fact  that  lactose  is 
inverted  in  the  body  indicates  its  presence  there.  Lactase  has 
been  found  in  the  foetal  intestinal  tract.  (Brochin.) 

Maltase,  the  third  of  the  prominent  inversion  ferments,  is 
very  widely  distributed  in  nature,  being  an  almost  invariable 
accompaniment  of  amylase  in  plants,  yeasts,  and  bacteria,  and  ex- 
isting also  in  some  lower  forms  of  vegetable  life  that  possess  no 
amylase.  Maltose  is  present  in  the  salivary  secretion,  the  pan- 
creatic juice,  the  succus  entericus,  liver  tissue,  the  blood,  and  thus 
in  all  organs  of  the  mammalian  body.  That  a  particular  yeast 
almost  never  contains  both  lactase  and  maltase,  as  pointed  out  by 
Fischer,  is  a  good  illustration  of  adaptation  to  the  external  me- 
dium. 

The  reaction  of  inversion.  The  hydrolysis  of  the  disaccha- 
rides  is  a  direct  addition  of  water  and  cleavage  into  the  two 
components. 


170  University  of  California  Publications.      [PATHOLOGY 

Sacchrose     +     water    :        d-glucose     4-     d-laevulose. 
Maltose        +     water    =     d-glucose     +     d-glucose. 
Lactose        +     water    -   -    d-glucose     +     d-galactose. 

For  cane  sugar  the  reaction  would  run : 

CH.CHOH.CHOH.CH.CHOH.CH2OH  (  CHO(CHOH)4CH,OH 

+  H2O  =  < 
CH2.CU.CHQH.CHOH.COH.CH2OH  (  CH2OH(CHOH):)CO.CH,OH 

So  far  as  we  know  there  are  no  sub-stages  in  the  reaction.  The 
acid  hydrolysis  yields  the  identical  products,  and  there  are  no 
known  qualitative  differences  in  the  reactions.  The  hydrolysis 
occurs  in  pure  water,  though  very  slowly,  rapidly  in  steam.  This 
auto-hydrolysis  has  been  studied  recently  by  Lindet.  Solutions 
of  pure  cane  sugar,  and  of  the  two  products,  conduct  a  current 
somewhat  better  than  the  same  water  in  which  they  were  dis- 
solved, and  from  this  Lindet  infers  a  slightly  acid  character  in 
sugars,  or  in  betters  terms,  a  slight  dissociation.  Experiments  in 
glass  he  found  negative  on  account  of  the  alkali  from  the  glass ; 
the  different  metals  affected  the  hydrolysis  in  different  degrees, 
some  depressing,  some  accelerating,  while  some  were  of  no  effect. 
The  auto-hydrolysis  was  accelerated  by  the  addition  of  the  two 
hexoses  that  represent  the  products.  The  actual  agent  in  this 
auto-hydrolysis,  of  which  every  inversion  must  be  looked  upon 
as  the  acceleration,  is  to  be  sought  in  the  hydrogen  ion  of  the 
dissociated  water.  According  to  the  conception  of  catalytic  re- 
actions that  Euler  has  endeavored  to  introduce  into  the  world 
of  organic  chemistry,  the  sugar  must  also  be  looked  upon  as  sub- 
ject to  a  slight  degree  of  electrolytic  dissociation.  This  dissocia- 
tion may  be  conceivably  either  into  a  dextrose-ion  and  a  laevulose- 
ion,  or  into  a  cation  O^H^iOio4  and  an  anion  OH~.  Accord- 
ing to  such  a  conception,  the  conditions  in  a  solution  of  sugar  in 
water  correspond  to  a  system  of  four  active  ions  in  very  high 
dilution.  Euler  is  inclined  to  consider  that  the  more  probable  of 
the  two  possible  dissociations  is  the  latter,  and  that  the  hydrolysis 
is  determined  by  the  concentration  of  the  saccharose  cation. 


VOL.  1]  Taylor — On  Fermentation.  171 

It  must  be  clearly  understood  that  the  hypothesis  of  the  ionic 
dissociation  of  cane  sugar  is  practically  devoid  of  experimental 
basis,  and  represents  little  else  than  the  expansion  of  the  idea 
that  all  reactions  are  ion  reactions  to  the  domain  of  organic  chem- 
istry. For  the  practical  study  of  the  complicated  problem  of  the 
catalytic  acceleration  of  the  hydrolysis  of  a  sugar,  the  hypothesis 
presents  no  obvious  advantage.  It  has  up  to  the  present  been 
impossible  to  demonstrate  ionic  dissociation  in  the  vast  number 
of  organic  substances  in  the  sense  in  which  the  law  of  Arrhenius 
may  be  shown  to  hold  for  inorganic  substances.  That  all  reac- 
tions, in  whatever  medium  they  occur,  are  ion  reactions,  cannot 
be  maintained  today.  The  experimental  problem  in  the  fermen- 
tations is  already  difficult  enough,  and  it  is  neither  simplified  nor 
elucidated  by  the  introduction  of  the  hypothesis  of  Euler. 

The  members  of  the  platinum  group  all  act  as  accelerators  to 
the  acid  inversion  of  sugar.  They  accelerate  the  auto-hydrolysis 
as  well  as  the  acid  hydrolysis,  acting  for  the  latter  especially  as 
zymoexciters  rather  than  as  additional  catalysors.  Plzak  and 
Husek  have  shown  that  for  the  members  of  the  whole  platinum 
group  zinc  and  manganese  act  as  negative  catalysors.  I  have  de- 
termined that  platinum  is  not  a  zymoexciter  for  invertase,  and  if 
it  act  at  all  as  an  accelerator  under  such  circumstances  that  ac- 
tion is  so  weak  as  not  to  appear  in  the  final  results. 

In  addition  to  its  faculty  of  acceleration  of  the  hydrolysis  of 
cane  sugar,  invertase  possesses  the  faculty  of  accelerating  the 
hydrolysis  of  certain  complex  sugars.  Gentianose,  a  complex 
carbohydrate  existing  in  Gentiana  lutea,  consists  of  two  mole- 
cules of  d-glucose  and  one  molecule  of  d-laevulose.  Invertase 
has  the  faculty  of  splitting  off  one  molecule,  that  of  d-laevulose, 
leaving  a  disaccharide  remaining,  gentiobiose,  composed  of  two 
molecules  of  d-glucose.  This  gentiobiose,  though  composed  of 
two  molecules  of  d-glucose,  is  not  identical  with  maltose  in  its 
chemical  and  physical  properties,  and  is  not  fermentable  by  mal- 
tase,  but  is  fermentable  by  emulsin.  In  a  similar  manner  inver- 
tase is  able  to  accelerate  the  partial  hydrolysis  of  another  com- 
plex sugar,  melitriose,  a  carbohydrate  composed  of  a  molecule 
each  of  d-glucose,  d-galactose,  and  d-laevulose.  Invertase  will 
ferment  melitriose  in  the  sense  that  it  will  split  off  the  molecule 


172  University  of  California  Publications.      [PATHOLOGY 

of  d-laevulose,  leaving  a  disaccharide,  melibiose.  This  melibiose 
is  composed  of  one  molecule  of  d-galactose  and  one  of  d-glucose, 
but  it  is  not  identical  with  milk  sugar;  it  has  different  physical 
and  chemical  properties;  it  is  not  fermentable  by  lactase,  but  is 
fermentable  by  emulsin  and  by  a  ferment  present  in  certain 
yeasts,  that  again  will  not  ferment  milk  sugar.  (Bourquelot, 
Fischer.)  Invertase  is  able,  furthermore,  to  accelerate  the  hy- 
drolysis of  the  synthetic  a-methyl-fructosides  of  Fischer  (not 
the  &-fructosides),  though  unable  to  influence  the  a-methyl  glu- 
cosides  of  d-glucose  or  d-galactose.  These  several  facts  seem  to 
ally  the  acceleration-faculty  of  invertase  to  the  presence  of  d- 
laevulose  in  the  disaccharide.  This  is  of  especial  interest  when 
taken  in  connection  with  the  fact  that  of  the  products  of  the  re- 
action of  inversion  it  is  apparently  the  d-laevulose  alone  that 
exerts  the  function  of  the  law  of  mass  action  on  the  progress  of 
the  reaction  of  hydrolysis ;  if  the  d-laevulose  be  removed  from 
the  system,  the  d-glucose  does  not  depress  the  progression,  while, 
on  the  other  hand,  the  removal  of  the  d-glucose  alone  is  entirely 
without  effect.  This  suggests  apparently  a  fundamental  distinc- 
tion between  the  accelerations  of  acid  and  invertase  on  the  hy- 
drolysis of  sugar.  The  distinction  may,  however,  be  only  quan- 
titative, and  not  qualitative ;  exact  investigations  are  needed  here. 
Weak  acid  is  able  to  effect  only  the  first  stage  of  the  hydrolysis 
of  gentianose  and  melitriose,  the  splitting  off  of  the  d-laevulose, 
analogous  to  the  action  of  the  invertase ;  strong  concentrations  of 
acid  will,  however,  effect  a  complete  cleavage. 

In  an  analogous  manner  maltase  seems  to  act  up  the  a  series 
of  d-glucose,  while  lactase  acts  upon  the  b  series  of  d-glucose  and 
d-galactose. 

Kinetics  of  the  reaction.  A  peculiar  interest  attaches  to  the 
study  of  the  kinetics  of  the  fermentation  of  cane  sugar,  since  it 
was  with  this  body  that  Wilhelmy  first  established  the  law  of 
reaction  bearing  his  name.  It  has  naturally  attracted  much  at- 
tention, since  it  is  one  of  the  most  feasible  of  fermentations  with 
which  to  work ;  the  substrate  may  be  procured  in  a  state  of  high 
purity,  the  ferment  is  easily  obtained,  and  the  measurement  of 
the  reaction  with  the  polarimeter  is  simple  and  accurate. 

0 'Sullivan  and   Thompson  first  studied  the  question  in   a 


VOL.  ij  Taylor— On  Fermentation.  173 

quantitative  way.  They  believed  that  their  results  could  be  rep- 
resented by  a  logarythmal  curve,  but  a  closer  inspection  of  their 
figures  shows  that  there  is  a  gradual  increase  in  the  figures  for 
the  constants  as  the  reaction  progresses.  Tammann  about  the 
same  time  showed  that  the  reaction  with  invertase  is  not  a  com- 
plete reaction  as  in  the  case  of  an  ordinary  acid  inversion. 
While  his  figures  indicate  in  general  a  rough  conformation  to 
the  law  of  acid  inversion,  there  were  many  deviations.  These 
Tammann  sought  to  attribute  in  part  to  the  differences  in  the 
relations  with  varying  amounts  of  ferments;  with  high  concen- 
trations of  ferment  the  reaction  seemed  to  lag  behind ;  with  lower 
concentrations  it  seemed  to  rush  ahead. 

Duclaux  next  studied  fermentative  inversion.  He  began  the 
study  with  several  preconceptions  that  seriously  influenced  his 
work,  but  in  other  ways  his  methods  were  so  superior  to  those  of 
the  contemporaneous  biological  world  that  he  is  to  be  regarded 
as  the  father  of  accurate  biological  study  of  fermentation.  He 
employed  in  large  part  high  concentrations,  and  although  in  his 
tests  with  low  concentrations  the  velocity  was  proportional  to 
the  mass  of  the  substrate,  the  fact  that  at  high  concentrations 
the  reaction  seemed  independent  of  the  mass  of  substrate  led  him 
to  reject  the  doctrine  of  mass  action.  He  held  that  in  the  begin- 
ning the  reaction  exhibited  a  regular  progression,  that  the  quan- 
tity of  substance  transformed  was  proportional  to  the  time, 

at 

=  C  (thus  corresponding  to  a  straight  line),  and  that  this  con- 
dition persisted  until  about  one-fifth  of  the  sugar  was  inverted, 
when  the  reaction  became  lessened  owing  to  the  influence  of  the 

products  of  reaction.    From  this  point  on  he  regarded  the  reac- 

/  A 

tiou  as  being  represented  by  the  equation  C-  -  ~  log  A_x  .     He 

studied  in  detail  the  retardation  of  the  products  upon  the  pro- 
gress of  the  reaction.  This  retardation  he  conceived  as  depending 
not  upon  the  mass  of  the  products  themselves,  but  in  their  rela- 
tion to  the  initial  concentration  of  the  substrate,  and  this  relation 
he  defined  for  each  moment  in  the  reaction  to  be  —  .  Obviously 
this  manner  of  conception  is  entirely  out  of  harmony  with  the 
law  of  mass  action,  as  well  as  with  the  kinetic  considerations  con- 
cerned in  a  reaction  of  reversibility,  and  indeed  Duclaux  specifi- 


174  University  of  California  Publications.      [PATHOLOGY 

cally  rejected  the  possibility  of  a  reversion  of  ferment  action 
upon  our  present  basis  of  thermodynamics. 

A.  N.  Brown  confirmed  the  previous  statements  that  in  high 
concentrations  a  constant  unit  of  transformation  occurs  in  a  unit 
of  time,  and  that  later  the  results  proceed  with  greater  rapidity 
than  could  be  accounted  for  in  a  simple  logarythmal  curve.  He 
also  confirmed  the  observations  of  Duclaux  upon  the  retardative 
action  of  the  products  of  fermentation. 

Within  recent  years  the  kinetics  of  the  reaction  of  inversion 
of  cane  sugar  have  been  thoroughly  worked  through  by  Victor 
Henri.4  He  employed  pure  substances  and  worked  under  care- 
fully controlled  conditions  of  concentration,  temperature,  etc. ; 
and  most  of  all,  he  proceeded  upon  an  entirely  clear  definition 

of  the  actual  problem.    The  figures  he  obtained  for  the  constants 

1  A 

according  to  the  equation  C  =  -r  log  -. —  exhibited  a  regular 

v  ^.  —  'X 

and  progressive  numerical  increase.  Thus  in  four  different  ex- 
periments the  figures  for  the  constants  rose  from  25  to  33,  57  to 
88,  58  to  96,  and  from  105  to  185,  increases  more  regular  and 
pronounced  than  had  been  previously  observed.  Employing  the 
Ostwald  method  for  the  calculation  of  a  catalytic  auto-catalysis, 

Henri   obtained   an   empiric   equation,   which  when   integrated 

1      A  -\-  x 
reads:  2  &I  =  -T  1  -,    —  .     When  calculated  with  the  aid  of  this 

t         A.  —  X 

equation,  the  new  constants  exhibited  a  satisfactory  uniformity. 
Henri  further  confirmed  the  observations  that  invertase  is  not 
appreciably  inactivated  during  the  course  of  a  fermentation  of- 
moderate  length.  This  he  accomplished  by  adding  new  sugar  to 
the  system,  and  observing  that  on  the  restoration  of  the  original 
concentration  the  original  velocity  of  the  reaction  was  restored. 
He  found  that  with  dilute  solutions  the  reaction  was  in  general 
terms  proportional  to  the  mass  of  substrate,  though  this  held  but 
to  quarter  normal  solutions.  Nevertheless  the  velocity  of  the 
reaction  in  the  favorably  dilute  solutions  did  not  hold  pace  ex- 
actly with  the  substrate  concentration,  but  depended  in  part  upon 
the  concentration  of  the  products.  As  a  rule  he  found  that  the 
lower  the  concentration  the  higher  the  constant.  The  accelera- 
tion of  the  reaction  he  found  quite  closely  proportional  to  the 
quantity  of  ferment. 


VOL.  l]  Taylor — On  Fermentation.  175 

1         A.  I  x 
This    empiric    equation    of    Henri  —  2  K  =  —    1    -   ~    was 

of  very  doubtful  value.  In  the  first  place,  while  the  constants 
obtained  through  its  use  exhibited  fairly  uniform  concordance 
in  a  particular  experiment,  they  were  not  at  all  concordant  in 
different  tests  with  different  substrate  concentrations.  They  ex- 
hibited in  short  the  same  relations  that  were  described  for  the 
constants  of  the  amylase  reactions.  Secondly,  it  is  certain  that 
there  is  not  an  auto-catalysis  in  the  inversion  experiment;  the 
products  depress,  they  do  not  stimulate  the  reaction  of  the  fer- 
mentative acceleration.  That  the  constants  increase  as  the  con- 
centration of  the  products  increase  is  true,  but  this  is  not  to  be 
interpreted  to  indicate  an  auto-catalysis,  contradictory  as  this 
statement  may  seem  to  be,  because  the  direct  experiment  shows 
the  products  to  be  negative.  The  facts  suggested  that  the  in- 
crease in  the  constants  was  associated  with  some  alteration  in  the 
intensity  of  the  enzymic  action ;  the  enzymic  acceleration  was  in- 
terpreted to  depend  not  strictly  on  the  ferment  concentration, 
but  also  on  the  substrate  and  product  concentrations.  Henri  next 
attempted  to  define  these  relations,  and  his  studies  on  this  point 
constitute  really  the  first  solid  beginnings  of  a  theory  of  the  dyn- 
amics of  fermentation. 

Henri  based  his  theories  on  the  assumption  that  the  catalytic 
and  fermentative  accelerations  rest  chemically  on  intermediary 
reactions.  The  ferment  may  be  obviously  in  combination  with 
the  substrate,  with  the  products  or  free.  It  is  assumed  by  Henri 
that  all  three  states  exist;  that  the  combinations  with  the  sub- 
strate and  products  are  not  complete  and  are  subject  to  the  law 
of  mass  action.  Obviously  therefore,  as  the  fermentation  pro- 
ceeds the  relation  of  the  combinations  with  the  substrate  and  the 
products  respectively  is  being  constantly  shifted.  Since  these 
considerations  promise  to  be  of  much  importance  to  the  future 
developments  of  fermentations,  we  shall  consider  them  in  detail, 
the  more  as  the  brochure  of  Henri5  is  difficult  of  access. 

In  the  chemical  system  under  consideration,  A  is  the  sub- 
strate, the  sacchrose ;  F  is  the  ferment,  x  the  sugar  inverted  in 
the  time  t,  and  A-x  the  amount  of  substrate  in  the  system  at  the 
end  of  the  time  t.  Under  the  assumption  that  the  ferment  F  is 
divided  into  three  parts,  that  combined  with  the  substrate  we  will 


176  University  of  California  Publications.      [PATHOLOGY 

term  Fs,  that  with  the  products  we  will  term  Fp,  and  the  free  fer- 
ment we  will  term  Ff.  These  combinations  are  to  be  considered 
in  equilibrium,  in  accordance  with  the  law  of  mass  action.  The 
total  ferment  mass  is  then  F  =  Fs  -\-  Fp  -\-  Ff.  The  equilib- 
rium between  the  ferment  and  the  substrate  is  expressed  in  the 
equation:  Ff  (A  —  x}  =  —  Fs.  The  equilibrium  between  the 
ferment  and  the  products  is  expressed  in  the  equation :  Ff  x  = 
-  Fp.  m  and  n  are  the  constants  of  equilibrium.  The  amount 

of  ferment  thus  free  and  combined  may  be  then  calculated.    Thus 

F  m.  F.  (A—x)  . 

Ff=  T    — 7~4 — r~i —  and  Fs  =  —  —  .    The  acceleration 

3        1  +  m  (A — Of)  -+-  nx  1  -\-  m  (A — .r)  +  nx 

may  be  related  either  to  the  ferment  combined  with  the 
substrate  (Fs}  or  to  the  ferment  free  in  the  system  (Ff}.  If 
the  acceleration  be  related  to  the  ferment  in  combination  with 
the  substrate  (Fs},  the  differential  equation  representing  the  re- 
action would  be  — -^ -—  C.  Fs;  replacing  now  the  Fs  with  the  equa- 

dx  C.  m.  F.  (A—x) 

turn  for  it  as  given  above,  we  have:  — ^  =  ^  +  m(A_x)+nx-   if,  on 

the  other  hand,  the  acceleration  be  related  to  the  ferment  free  in 
the  system,  it- would  be  proportional  to  the  free  ferment  and  to 
the  substrate,  and  the  differential  equation  to  express  this  rela- 
tion would  be  -—==(7.  Ff.  (A—x}  •  when  the  value  of  Ff  as 

dx  C.  F.  (A—x) 

given  above  is  inserted,  we  have:  — ~dJ~  i  +  m(A—x\-^nx  •     inese 

two  equations  are  identical;  it  is  therefore  immaterial  to  the 
mathematical  development  of  the  situation  whether  we  assume  the 
active  mass  of  the  ferment  to  be  that  related  to  the  substrate  or 
free  in  the  system.  The  considerations  based  on  the  equilibria 
between  these  several  relations  leads  us  then  to  substitute  for  the 

dx          ,    A  dx  G's  (A—x) 

simple  equation  fU  =  l/^— the  equation  —  dt  —  1+m(A—X)  +  nxi  m 


which  C3  is  a  constant  proportional  to  the  quantity  of  ferment, 
m  and  n  constants  that  characterize  the  relations  of  the  ferment 
to  the  substrate  and  products  respectively  (they  will  vary  with 
alterations  in  the  medium  and  with  temperature),  A  is  the  ini- 
tial concentration  of  substrate  expressed  in  normal  terms,  x  is 
the  substrate  inverted  in  the  time  t.  For  25°,  with  solution  of 
not  over  half  normal,  Henri  found  m  and  n  to  be  respectively 
30  and  10.  When  the  above  equation  is  integrated  under  the 


VOL.  1]  Taylor — On  Fermentation.  Ill 

assumption  that  when  /  =  0,  x  =  0,  we  have:   Cs  =  ^[(>« — w) 
;  +  H.  1  -        I  +  —  1 1,    -  .     When  the  values  for  m   (30)  and  n 

A  A       .t  f        .^1 3? 

^0  .4  r      'X 
(10)  are  inserted  into  the  equation,  we  have:    €3  =  — ' :~~|_2~7 

A     -i         1        A 
\- 1  -7  7-  1  7  — .     This   is   the    equation   employed    in  the 

J3L ./  t         ^i        •/ 

calculations.  The  equation  assumes,  apart  from  strictly  con- 
trolled conditions  of  experimentation,  the  non-in activation  of 
the  ferment,  the  non-reversion  of  the  reaction,  and  the 
absence  of  auto-cat alysors.  The  drawback  of  the  equation 
lies  in  the  presence  of  three  constants;  equations  with  three 
constants  approach  closely  interpolation  equations;  the  m 
and  n  ought  to  be  determinable  experimentally,  or  at  least 
controllable.  The  values  given  for  m  and  n  are  not  accurate, 
only  approximate.  One  can  see  from  the  figures  of  Henri  that 
the  agreement  in  the  constants  would  be  closer  were  m  relatively 
somewhat  greater.  With  this  equation  Henri  has  calculated  the 
results  of  a  large  number  of  experimental  findings.  The  Cs  is 
fairly  concordant  in  the  same  series,  and  also,  what  is  of  more 
importance,  closely  concordant  in  different  series,  with  varying 
substrate  concentrations.  The  equation  therefore  appears  to  so 
closely  express  the  intensity  of  ferment  activity  that  taken  into 
connection  with  the  relations  of  concentration  in  the  system,  re- 
sults are  obtained  that  are  in  close  accord  with  the  law  of  mass 
action.  The  equation  of  Henri  is  in  reality  nothing  else  than 

n 

his  equation  for  the  intensity  of  ferment  action  1=  -1- 

1  -\-  m  (A — x)  -f-  nx 

inserted  into  the  equation  for  a  monomolecular  reaction.  Henri 
studied  maltase,  and  found  the  constants  to  exhibit  a  curve ;  first 
they  rose  and  later  fell.  There  was,  however,  no  regularity  in  the 
curve. 

Barendrecht  in  his  recent  paper  attempting  to  explain  the 
nature  of  fermentation  as  the  result  of  emanations,  reported 
upon  experiments  with  invertase.  His  figures  quite  closely  re- 
semble those  of  Henri. 

Visser  has  recently  published  a  study  of  the  reaction  of  fer- 
mentation based  on  the  hypothesis  that  the  deviation  in  the 
results  from  the  common  law  is  due  to  variations  in  the  intensity 
of  the  ferment  action,  and  this  he  tries  to  relate  to  the  concen- 


178  University  of  California  Publications.      [PATHOLOGY 

trations  in  the  system  in  accordance  with  the  law  of  mass  action. 
He  first  attempted  to  include  in  his  considerations  tli<>  l';iH  Hint 
the  reactions  of  fermentation  are  reversible,  and  grounded  an 
equation  on  the  relations  of  the  constants  of  equilibrium.  The 
reaction  he  represented  in  the  equation  —  '  '  =  C\  ( A  -  -  .'• ) 
C2.x2.  When  this  is  integrated  under  the  ;i.ssumplion  tli.-il  when 

t  =  0,  x  =  0,  we  have  :  Const.  =  4  1  (A~"\    /  (^~^~?l  •  &  is  the 

t      (A— a)     (  (A—x)—l>) 

concentration  of  the  substrate  in  the  stage  of  equilibrium,  and  a 
may  be  calculated  when  b  is  known  by  means  of  ab  =  A2.  These 
constants  did  not  give  good  agreement  for  the  experimental  re- 
sults. A  consideration  of  the  facts  will  indicate  that  this  could 
not  have  been  expected.  While  it  is  true  in  the  theoretical  sense 
that  the  reaction  of  inversion  is  reversible,  in  practice  it  is  not 
so  because  the  station  of  equilibrium  is  so  near  the  point  of  a 
complete  reaction.  When  a  solution  such  as  is  used  in  inversion 
experiments  is  in  equilibrium,  we  have  sacchrose  1  :  products 
99.  Obviously  therefore  the  constant  for  the  reaction  in  the 
direction  of  the  left,  the  <—0  in  the  equation,  must  be  very  small 
in  comparison  to  the  constant  for  the  reaction  in  the  direction 
of  the  right,  the  C->  of  the  equation .  Under  these  circumstances, 
the  equation  as  a  matter  of  fact  is  almost  identical  in  its  mathe- 
matical valuation  to  the  common  equation  for  a  monomolecular 
reaction.  This  last  equation,  however,  it  will  be  recalled,  does 
not  give  concordant  constants;  the  figures  rise  progressively.  So 
do  they  rise  progressively  if  the  equation  of  Visser  is  used.  Fur- 
thermore, the  apparent  station  of  equilibrium  cannot  in  our 
present  state  of  knowledge  be  entirely  identified  with  the  station 
of  equilibrium  in  a  reaction  in  a  simple  system  like  ester  -{-water 
=  alcohol  -f-  fatty  acid.  Knoblauch  has  shown  that  the  constant 

/•! , 

of  equilibrium  =  '_£„  and  when  the  system  is  in  ;i  state  of  equi- 
librium the  transformation  in  the  one  direction  exactly  equals 
that  in  the  other.  This  is  in  direct  accord  with  the  theory.  Now 
when  in  an  inversion  experiment  the  reaction  ceases  in  the  situ- 
ation sugar  1  <=>  products  99,  it  is  to  be  assumed  in  accord- 
ance with  the  theory  that  in  each  moment  as  much  sugar  is  being 
reformed  as  there  is  sugar  being  hydrolyzed.  But  it  is  possible  to 
show  experimentally  that  this  is  not  the  case.  Experiments  in  the 


V<" ..  I  I  Tiii/lor-      (hi    !''<  nn>  iilalinn.  .     179 

reversion  of  ferment  re;idions  iiidicnle  positively  lluil  il  will  re- 
(|iiire  months  to  reform  the  iiiiioiint  of  simnr  th;it  m;iy  under  the 
s;ime  conditions  lie  li\  droly/ed  within  ;i  l'e\v  hours.  While  the 
tlieoretieiil  c(|ii  i  I  ihr  i  ii  m  is  without  doubl  conhiined  in  the  ;ippar- 
ent  e(|iiilil»rinm  oliserved.  some  other  t'.-ietor  or  l';ietors  ;ire  ;ilso 
eont;iined  therein,  iind  one  of  these  f;ic|ors  is  iimpies!  ion;il>ly  re 
hiterl  to  the  rermeiil  ilsell'.  These  eoiisider;it  ions  indic;i1e  tluit 
the  use  of  the  theory  of  the  reversihility  of  tile  reaction  ol'  hy- 
drolysis in  the  e<|ii;ition  for  the  re;ietion  velocity  :is  |>ro|>o,sed  by 

Yisser    ( Id    not     llMVe    lieeil    expected    to    |e;u|    to    COnCOnhlll     re 

suits;  ;ind  the  pr;ie!ie;il   findings  ;ire  in   1'nll  ;iceord  herewith. 

Yisser  next    iilteinpted   to  define  the  intensity   of   rermeiil    M 
lion.     This  he  did   in  ;i   purely  empirical   iimnner.      Upon   the  ;is 
siini|)tion   th;it    the   intensity   of   rermeiil    jiclimi    is   rehiled   lo   the 
Concentration    <»!'    the   snhslnite    ;ind    of    the    products,    he    found: 

Ferment  intensity  (I)  ,,  (,  , '"  ((  r),.  This  lie  inserted 
Into  the  previous  equation  -  'f''u  -  <\  (A  —  .<•}<'•>.  .<•''.  Since  t  he  v.-dne 

of  this  ei|ii;i(ion  is  so  iiejirly  th;il  of  the  old  eqimlion.  I  he  l.-itler, 
heiiiM-  much  simpler,  m;i\  1 mployed.  This  will  yield  .-rr  =  (7 

(A-./-)/.      ;',;>,(.!-  ,•».  /(        n  /  (  when  inte- 

^r;ited  on  the  .-issiimpt  ion  Mint  ./•  (t  when  /  <>.  we  h;i\e:  '_'  (\ 
<'-.!  (-I  i  (-!-.'•))(.!-(,!-./•))  I  S.I  I  ('  .  Th<-  results 
iire  l';iirly.  conconhint  in  the  dit'l'eienl  me;isn  rements  of  the  s;ime 
series,  hnl  not  ;il  ;ill  concord;int  in  the  different  iiie;isiir<'iiieiil  of 
;it  vjiryin-j  con,  -ent  r;il  ions.  In  Ihis  respecl  they  ;ire  wilhoiil 
Ilie  \;didily  th;il  is  ;dt;iched  to  the  coiisl;inls  when  c;ilciil;ilei| 
••iccordiii"  In  I  lie  ei|ii;il  1011  of  I  lenri. 

ller/o,^   h;is   iipplied    to    inversion    the    N'ernsl    tlieory   of    iv.ie 
lion   velocily   in  ;i   hetero-eiieons  system.      Il    will   he  recjilled  th;il. 
in  the   Xeriisf.  present;il  ion  stress  is  hid   upon   the    |';K-|    lh;il    such 

slirriii";  must,  he   provided   lh;il    the   relations  of  concent  i-;it  i n 

the  film  of  hoiimhry  cont;iH  of  the  I  wo  pluses  provide  for  ;i 
very  thin  hyer  in  which  the  velocily  of  diffusion  then  beOOmei 
the  determining  \;in;iUe,  the  velocity  of  chemic;d  re;ii'lion  lieinv; 
nssiimed  U)  be  Of  practically  inlini!esim;d  velocil.y.  This  si  in m» 
must  he  such  ;is  to  ohliter;de  I  he  effects  of  I  In-  convect.iori 


180  University  of  California  Publications.      [PATHOLOUY 

in  a  word,  the  concentration  of  the  solution  in  the  system  must 
be  everywhere  the  same,  and  under  these  circumstances  the  only 
diffusion  velocity  variable,  and  therefore  measurable,  will  be 
located  at  the  boundary  of  contact  of  the  two  phases.  In  the 
application  of  this  proposition  to  a  system  in  a  state  of  fermenta- 
tion, Herzog  regards  the  heterogeneous  system  as  comparable  to 
a  capillary  system,  in  which  the  capillary  walls  are  formed  by 
the  surfaces  of  the  colloidal  particles  of  the  ferment  in  the  case 
of  the  inversion  of  sugar,  of  both  the  ferment  and  the  substrate, 
however,  in  the  case  of  the  fermentation  of  protein.  Variations 
in  density  will  result  in  the  setting  up  of  circulating  currents 
through  the  capillary  spaces.  The  velocity  of  this  circulating 
stream,  however,  will  depend  on  the  viscosity,  the  internal  fric- 
tion, of  the  system.  The  higher  the  viscosity,  the  slower  the  cur- 
rents ;  and  the  slower  the  currents,  the  less  the  unit  of  substrate 
that  will  be  carried  to  the  surface  of  the  ferment  in  the  unit  of 
time.  In  this  proposition  are  two  assumptions.  It  is  assumed 
firstly  that  the  surface  dimensions  of  the  colloid,  the  ferment, 
are  constant ;  that  the  dimensions  of  the  contact  of  the  two  phases 
are  constant ;  variations  in  this  are  recognized  by  Herzog  as  lead- 
ing necessarily  to  an  auto-catalysis.  It  is  assumed  secondly  that 
the  thickness  of  the  boundary  film  is  maintained  constant,  that 
the  capillary  currents  fully  realize  the  conditions  of  the  Nernst 
hypothesis.  These  being  granted,  the  reaction  velocity  (i.e.,  the 
diffusion  velocity)  may  be  taken  to  be  an  exponential  function 
of  the  viscosity,  and  an  equation  is  feasible.  This  reads:  C  = 

(•)    ,  in  which  n  in  the  function  of  the  viscosity  and  m   a 
M  / 

constant  that  may  be  determined  by  the  equation  of  Rudorf . 

The  viscosity  of  certain  solutions  may,  according  to  Rudorf, 
be  determined  from  the  concentrations  according  to  the  equation 
n  =  R  -j-  Aa  -+-  Ba2  -f-  Ca3,  or  better  under  the  proposition  that 
the  viscosity  of  water  =  1:  n  =  A-{-Ba-\-  Ca2  -f  Das.  n  is  the 
internal  friction,  A  the  viscosity  of  water  (1),  a  the  concentra- 
tion, and  A,  B,  C,  and  D  constants.  On  the  assumption  that 

R=Q,  we  then  obtain  C==  (-Jjj+J*Tc*rT '      m  is  de' 

termined  by  calculation  to  be  one-half.     A,  B,  and  0  must  be 
determined  for  each  different  ferment.     The  results  of  the  cal- 


VOL.  l]  Taylor — On  Fermentation.  181 

culation  of  the  experimental  findings  are  fairly  satisfactory,  the 
constants  are  quite  concordant. 

Henri5  has  pointed  out  that  the  equation  of  Herzog  is  in  con- 
tradiction to  the  diffusion  law  of  Fick.  Herzog  assumes  R  =  0, 
which  would  need  to  be  demonstrated.  Henri  has  also  pointed 
out  that  the  actual  viscosities  of  the  sugar  solutions  are  not  at 
all  identical  with  the  postulated  viscosities  as  calculated  from 
the  constants  A,  B,  and  C.  To  my  mind,  the  most  direct  reflec- 
tion on  the  equation  of  Herzog  is  contained  in  the  fact  that  the 
addition  of  such  amounts  of  d-glucose  and  d-laevulose  as  possess 
the  same  internal  friction  should,  according  to  the  hypothesis, 
exert  the  same  influence  upon  the  reaction  velocity,  whereas  as 
a  matter  of  fact  d-laevulose  is  potent,  while  d-glucose  is  practi- 
cally without  influence.  The  velocities  of  different  fermentations 
vary  enormously  under  conditions  in  which  such  variations  in 
diffusion  cannot  be  assumed  to  occur.  Negative  to  the  idea  is 
also  the  fact  that  within  certain  limits  the  larger  the  proportion 
of  substrate  to  ferment  the  closer  the  theory  is  observed.  That 
trypsiii  displays  the  same  behavior  when  digesting  protamine, 
scarcely  colloidal,  as  when  digesting  the  coarse  suspensions  of 
coagulated  pulverized  egg  albumin,  is  also  not  to  the  favor  of 
the  Herzog  hypothesis.  Herzog  has  himself  pointed  out  that  an 
auto-catalysis  is  almost  inexplicable  under  this  theory.  The  ac- 
celeration of  velocity  by  increase  in  temperature  is  also  out  of 
harmony  with  the  diffusion  hypothesis.  Herzog  does  not  himself 
regard  his  equation  as  more  than  an  orientation  interpolation 
equation,  and  while  the  insufficiency  of  the  equation  as  repre- 
senting the  progression  of  a  fermentation  is  to  be  admitted,  the 
fact  remains  that  it  deals  with  an  aspect  of  the  problem,  the  con- 
dition of  the  heterogeneity  in  the  reacting  system,  that  had  been 
hitherto  overlooked.  And  while  it  may  be  admitted  that  the  dom- 
inant variable  may  not  be  located  in  the  relations  of  this  hetero- 
geneity, an  influential  variable  is  certainly  present  there.  This 
statement  is  illustrated  by  known  facts  in  the  domain  of  colloidal 
metals.  The  catalytic  action  of  colloidal  platinum,  for  example, 
is  not  a  function  of  the  mass  of  platinum,  but  rather  of  the  fine- 
ness of  its  subdivision  into  colloidal  particles.  Different  prepara- 
tions of  colloidal  platinum  of  the  same  mass  percentage  vary 


182  University  of  California  Publications.      [PATHOLOGY 

widely  in  the  colloidality  of  the  suspension ;  some  are  almost 
amorphous,  others  are  typically  colloidal.  Now  the  catalytic  ac- 
tivity of  such  suspensions  is  in  general  terms  proportional  to 
the  colloidality  of  the  preparation.  The  actual  acceleration  may 
be  confidently  assumed  to  lie  in  intermediary  reactions  in  which 
the  metal  acts  in  chemical  combination.  But  the  effectiveness  of 
these  intermediary  reactions  is  closely  allied  to  the  colloidality 
of  the  suspension  of  platinum. 

As  a  matter  of  fact,  the  theory  of  Nernst  ought  not  to  be 
judged  by  this  application,  the  provisional  nature  of  which  has 
been  emphasized  by  Herzog  himself.  The  equation  of  Rudorf 
was  not  designed  for  the  present  type  of  calculation.  Eudorf 
determined  the  relation  between  the  concentration  and  viscosity 
of  solutions  of  acetic  acid,  urea,  sugar,  tartaric  acid,  propyl  alco- 
hol, and  acetone  in  water.  He  found  that  neither  the  older  ex- 
ponential equation  of  Arrhenius  (n  =  Ax}  nor  the  linear  equa- 
tion (n  —  l-\-an)  corresponded  to  the  actual  relations.  With 
the  simple  solutions  employed  by  him  he  found  that  the  molecular 
relations  in  the  system  were  never  constant;  if  the  substance 
underwent  polymerization,  the  deviations  tended  to  be  propor- 
tional to  n ;  if  combination  occurred  between  solvent  and  solute, 
the  deviations  tended  to  be  proportional  to  n2.  If  an  electrolyte 
wrere  present  in  the  system  the  relations  would  be  very  complex, 
and  Rudorf  studied  no  such  system.  If  two  substances  were 
present,  the  relations  would  be  much  more  complex,  even  were 
they  both  undissociated,  and  Rudorf  studied  no  such  system.  He 
studied  solutions  of  urea,  sodium  bromide,  and  hydrochinon  in 
acetic  acid,  but  the  results  were  not  interpretable  under  the  equa- 
tion. Now  Herzog  takes  the  equation  that  was  erected  to  express 
the  relations  between  the  concentration  of  a  pure  organic  sub- 
stance in  wrater  and  the  viscosity  of  the  solution,  and  applies  it  to 
a  complex  system,  in  the  attempt  to  relate  its  internal  friction 
to  its  concentration.  The  fermentation  system  contains  several, 
indeed  many  substances,  both  organic,  dissociated,  and  electro- 
lytic. There  is  in  the  work  of  Rudorf  no  warrant  for  this  use 
of  the  equation ;  it  was  not  erected  with  such  conditions  in  view 
and  is  not  adapted  to  account  for  them.  Rudorf  gives  as  the 
causes  for  the  deviations  of  the  results  from  the  linear  equation 


VOL.  1J  Taylor — On  Fermentation.  -.      183 

the  following  factors  that  operate  in  the  viscosity  of  a  solution. 
a.  The  electrical  situation,  whether  dissociated  or  not.  b.  Loose 
combinations  between  solution  and  solute,  c.  Combinations  be- 
tween the  molecules  of  the  solute,  polymerization,  d.  Electro- 
striction,  resulting  from  the  charging  with  ions.  In  the  case  of 
a  fermentation  system,  we  would  have  in  addition  the  combina- 
tions between  the  different  components  in  the  system.  It  may 
be  objected  that  apart  from  the  substrate  and  the  water,  the  other 
components  in  a  fermentation  system,  ferment,  salts,  and  other 
coincidental  organic  substances,  are  present  in  so  small  an  amount 
as  not  to  disturb  seriously  the  relations  of  the  solute  and  the 
solvent.  To  this  it  must  be  replied  that  only  the  experiment  can 
answer  to  what  extent  deviations  are  produced.  Such  experi- 
ments were  not  carried  out  by  Rudorf,  such  conditions  were  not 
contemplated  by  him,  and  the  application  of  the  equation  to  con- 
ditions for  the  interpretation  of  which  it  was  not  devised  is  an 
improper  procedure.  The  work  of  Dunstan  illustrates  the  ano- 
malies in  the  viscosities  of  mixtures,  and  finally  Fawsitt  has  re- 
cently shown  that  some  of  the  viscosity  measurements  of  Rudorf 
were  inaccurate. 

Armstrong1  has  very  recently  carried  out  accurate  studies 
upon  the  fermentation  of  milk  sugar,  based  upon  the  same  prop- 
osition that  during  the  course  of  a  fermentation  there  are  com- 
binations between  the  ferment  and  the  components  of  the  reac- 
tion system.  He  too  found  that  when  the  substrate  was  in  high 
concentration  the  velocity  was  not  proportional  to  the  mass  of 
substrate,  though  it  could  be  made  so  by  the  employment  of  huge 
quantities  of  ferment.  Diluted  solutions,  on  the  contrary,  ex- 
hibited a  velocity  proportional  to  the  mass  of  milk  sugar.  In 
solutions  of  high  concentration  the  curve  was  for  a  short  time 
linear,  then  became  logarythmal,  and  towards  the  close  again 
very  irregular.  Armstrong  therefore  divides  the  reaction  into 
three  stages :  in  the  first  the  transformation  is  a  function  of  time, 
in  the  second  the  velocity  is  a  function  of  the  concentration  of 
the  substrate,  in  the  last  the  regular  progression  of  the  hydrolysis 
is  disturbed  by  the  action  of  the  concentrated  products.  With 
lower  initial  concentrations  only  periods  two  and  three  are  ob- 
served. The  equation  of  Henri  agreed  very  well  with  the  results, 


184  University  of  California  Publications.      [PATHOLOGY 

except  for  the  last  period.  The  products  of  the  reaction  were 
found  to  be  actively  depressant,  and  this  retardation  was  caused 
solely  by  the  d-galactose.  This  corresponds  directly  with  the 
analogous  findings  of  Henri  that  laevulose  alone  depresses  inver- 
tase.  This  depression  of  the  action  of  the  lactase  was  not  due  to 
its  destruction ;  on  the  contrary,  the  ferment  seemed  very  resist- 
ant to  hydrolysis. 

Armstrong2  also  studied  the  acid  hydrolysis  of  milk  sugar  in 
order  to  compare  it  with  the  fermentation.  He  found  very  anal- 
ogous conditions.  With  a  high  concentration  of  the  substrate 
and  a  low  concentration  of  the  acid  the  early  reaction  follows  a 
linear  course;  later  the  logarythmal  curve  appears.  At  lower 
substrate  concentrations  and  with  a  greater  acid  concentration 
the  curve  is  logarythmal  from  the  start,  except  that  the  progress 
is  somewhat  in  advance  of  the  theory.  The  velocity  is  very  much 
slower  than  for  the  ferment,  something  like  1000  X  1.  He  con- 
siders that  the  two  inversions  are  quite  alike,  and  that  the  differ- 
ences are  due  largely  if  not  entirely  to  the  greater  affinity  of  the 
ferment  for  the  sacchrose  as  contrasted  with  that  of  the  acid,  and 
to  the  colloidal  nature  of  the  ferment. 

Armstrong3  also  studied  the  inversion  of  cane  sugar,  and 
reached  results  that  were  in  general  in  harmony  with  those  of 
Henri. 

Relation  of  ferment  mass  to  acceleration  of  reaction.  In 
practically  all  the  published  studies  in  which  this  question  has 
been  investigated  it  has  been  found  that  the  acceleration  was 
proportional  to  the  mass  of  ferment  in  the  system.  This  is  well 
illustrated  in  the  figures  of  Henri  and  Visser.  It  holds  true  only 
for  dilute  solutions,  the  same  concentrations  of  the  substrate,  in 
fact,  that  yield  the  best  results  in  investigations  to  determine  the 
relation  of  velocity  of  transformation  to  the  mass  of  substrate. 
It  is  most  interesting  too  to  observe  that  it  is  only  within  this 
range  of  concentrations  that  the  law  for  increase  of  reaction  with 
increase  of  temperature  holds  good. 

Reversion  of  reaction.  The  transformation  of  sacchrose  into 
the  hexoses  is  never  completely  accomplished  by  invertase.  In 
weak  solutions,  such  as  are  commonly  employed  in  the  investi- 
gations, the  untransformed  sugar  does  not  amount  to  over  1  or  2 


VOL.  1]  Taylor — On  Fermentation.  185 

per  cent.  In  more  concentrated  solutions  the  unconverted  por- 
tion may  be  as  high  as  5  or  more  per  cent.  While  therefore  the  ac- 
tual amount  left  unconverted  is  small,  it  may  be  easily  measured 
on  account  of  the  delicacy  of  the  polariscopic  method  employed. 
It  is  possible  therefore  to  show  that  the  addition  of  further  sugar 
after  the  reaction  has  passed  into  a  state  of  equilibrium  will  re- 
sult in  the  augmentation  of  the  inversion  until  the  state  of  equi- 
librium has  been  again  established.  If,  on  the  other  hand,  the 
products  be  removed,  the  remaining  substrate  will  be  for  meas- 
urable purposes  completely  inverted.  That  the  active  agent  in 
the  reversed  reaction  is  the  mass  of  d-laevulose,  and  not  the  mass 
of  d-glucose,  was  first  demonstrated  by  Henri,  and  it  is  easy  of 
confirmation.  This  is  clearly  shown  in  the  following  experiment. 
Four  systems  are  prepared.  In  a  we  have  a  standard  solution 
of  sugar  and  ferment ;  in  b  the  same  plus  a  unit  of  d-laevulose ; 
in  c  the  same  plus  a  corresponding  unit  of  d-glucose;  in  d  the 
same  standard  solution  plus  an  amount  of  invert  sugar  contain- 
ing the  d-laevulose  of  b.  The  sugars  must  be  pure  in  the  sterio- 
isomeric  sense.  The  results  of  the  experiment  are  that  the  veloc- 
ities of  a  and  c  are  alike,  the  velocities  of  b  and  d  alike,  and  much 
lower  than  a  and  c.  Henri  has  further  determined  that  the  in- 
hibitory action  of  the  d-laevulose  is  approximately  proportional 
to  its  mass  if  the  substrate  concentration  is  constant;  that  with 
a  fixed  mass  of  invert  sugar  the  depression  is  in  general  inversely 
proportional  to  the  concentration  of  the  substrate ;  and  that  when 
both  are  fixed  with  relation  to  each  other  the  retardation  is  pro- 
portional to  the  dilution  of  the  system.  These  facts  are  of  especial 
interest.  Not  only  does  the  actual  hydrolysis  of  the  cane  sugar 
under  the  acceleration  of  the  invertase  vary  with  different  sub- 
strate concentrations  disproportionately  to  the  law,  but  an  ad- 
ventitious variable  varies  in  its  disturbing  action  with  changes  in 
the  concentrations. 

Reversion  of  enzymic  activity  has  been  accomplished  for  the 
inversion  of  sugar ;  it  was  here  in  fact  first  accomplished.  Wohl 
and  Fischer  first  determined  that  when  sulphuric  acid  is  al- 
lowed to  act  upon  concentrated  solutions  of  glucose  a  disaccha- 
ride  was  formed  which  they  were  able  to  identify  as  isomaltose. 
This  is  obviously  the  converse  of  the  common  result  that  acid 


186  University  of  California  Publications.      [PATHOLOGY 

will  not  entirely  invert  concentrated  solutions  of  saccharose. 
Hill  attempted  the  reversion  by  the  use  of  a  yeast  that  was 
incapable  of  fermenting  glucose.  After  this  yeast  had  been  for 
some  time  in  contact  with  a  concentrated  solution  of  glucose,  the 
solution  was  found  to  have  acquired  an  increase  of  optical  rota- 
tion and  to  have  suffered  a  loss  in  the  power  of  reduction;  and 
from  the  solution  an  osazone  was  isolated  having  the  behavior  of 
maltosazone.  Emmerling1  repeated  the  experiment  and  obtained 
a  disaccharide  that  he  described  as  isomaltose.  Later  Fischer 
and  Armstrong  successfully  reversed  the  process  of  inversion 
of  milk  sugar.  They  allowed  a  lactase  from  the  kephir  yeast  to 
act  upon  solution  containing  d-glucose  and  d-galactose,  and  iso- 
lated a  disaccharide  which  they  identified  as  isolactose.  The 
reason  for  the  appearance  of  the  disaccharide  in  the  form  of  the 
isomeric  iso-sugar  has  not  been  investigated;  in  any  event  the 
question  is  of  qualitative  interest  solely.  From  the  standpoint 
of  the  relations  of  fermentation  to  the  steroisomerism  of  sugars, 
the  formation  of  the  iso-disaccharide  on  the  acceleration  of  the 
reversed  reaction  by  ferments  is  of  the  greatest  interest,  but  it 
in  no  way  qualifies  the  validity  of  the  fact  that  the  formation  of 
the  disaccharide  is  accomplished  under  the  influence  of  a  fer- 
ment. Armstrong4  has  investigated  in  detail  the  products  of  the 
reversed  action  of  ferments  on  the  synthesis  of  disaccharide  from 
maltose.  On  the  basis  of  the  well  known  work  of  Fischer  and 
himself  on  the  configuration  of  the  glucosides,  Armstrong  regards 
maltose  as  glucose-a-glucoside  and  isomaltose  as  glucose-6-gluco- 
side.  When  maltase  is  allowed  to  act  upon  a  solution  of  glucose, 
as  had  been  previously  known,  isomaltose  is  produced.  AVhen 
emulsine  is  allowed  to  act  upon  a  similar  solution  of  glucose,  mal- 
tose is  formed.  The  specificity  in  the  relations  of  the  two  fer- 
ments to  the  two  forms  of  glucoside  is  therefore  maintained  in 
the  syntheses.  When  the  synthesis  was  accelerated  by  the  pres- 
ence of  hydrochloric  acid,  both  maltose  and  isomaltose  were  deter- 
mined to  be  present.  Fischer  and  Armstrong  were  able  further 
to  show  that  lactase  could  condense  two  molecules  of  d-glucose  to 
a  biohexose  of  undetermined  nature.  Wroblowski  has  reported 
the  synthesis  of  cane  sugar  from  the  component  hexoses  under  the 
influence  of  invertase.  Finally  the  pancreatic  amylase  has  been 


VoL-  !]  Taylor — On  Fermentation.  187 

shown  by  Acree  and  Hinkins  to  be  capable  of  accelerating  the 
combination  of  acetic  acid  and  d-glucose  to  the  synthetic  tri- 
acetyl-glucose.  The  velocity  of  these  various  accelerations  of  the 
reversed  reactions  is  very  slow;  weeks  are  required  to  synthesize 
an  amount  of  the  disaccharide  that  can  be  hydrolyzed  within  a 
few  moments.  Since  these  reversions  are  naturally  supposed  to 
occur  in  nature,  it  is  evident  that  the  conditions  in  vivo  possess 
certain  relations  that  are  lacking  in  the  experiments  in  vitro. 

Relations  of  total  concentration  to  progress  of  reaction.  The 
work  of  Henri  has  illustrated  explicitly  what  had  been  approxi- 
mately indicated  in  the  earlier  work,  that  the  relation  between 
the  concentration  of  the  substrate  and  the  velocity  of  the  trans- 
formation holds  only  for  dilute  solutions.  The  most  favorable 
concentrations  are  from  0.1  to  0.5  normal  solution.  This  is  in 
marked  contrast  to  the  relations  in  the  inversions  by  acids,  which 
obey  the  law  under  much  wider  limits.  For  the  acid  hydrolysis, 
however,  the  concentration  of  substrate  may  be  reached  at  which 
the  law  is  no  longer  obeyed.  We  have  here  therefore  a  quanti- 
tative, not  a  qualitative  difference ;  the  operations  of  the  enzymic 
acceleration  of  the  inversion  follow  the  law  within  narrower  lim- 
its of  variations  in  the  substrate  than  in  the  case  of  the  acid 
acceleration. 

Influence  of  temperature.  In  a,  number  of  careful  investiga- 
tions the  activity  of  invertase  has  been  shown  to  increase  up  to 
35°  in  a  quite  strict  conformity  to  the  law  for  the  increase  of 
reactions  for  increases  in  temperature,  but  from  35°  to  55°  the 
increment  is  disproportionately  greater,  and  above  55°  the  power 
of  inversion  is  rapidly  destroyed  with  increasing  temperature. 
There  are  wide  variations  for  different  preparations,  and  this  is 
equally  true  for  maltase,  whose  optimum  temperature  seems  to 
be  in  general  some  15°  lower  than  for  invertase.  The  optimum 
temperature  does  not  seem  to  be  the  same  for  systems  of  high 
and  low  concentrations  of  substrate  and  ferment.  Invertase 
when  dry  is  very  resistant  to  heat,  and  may  be  heated  to  over 
100° ;  maltase  is  less  resistant.  When  dried  both  ferments  pre- 
serve their  activity  over  a  long  time.  Light,  in  the  presence  of 
free  oxygen,  is  destructive  to  invertase ;  otherwise  not. 

For  both  invertase  and  maltase  a  faintly  acid  reaction  seems 


183  University  of  California  Publications.      [PATHOLOGY 

advantageous,  although  at  a  neutral  reaction  fermentation  is  still 
very  active.  Alkalies  are  harmful  in  the  highest  dilutions.  The 
susceptibility  to  these  influences,  however,  varies  widely  with 
different  preparations;  some  do  best  in  a  1/300  normal  acid, 
others  in  one  ten  times  more  diluted.  The  other  conditions,  espe- 
cially temperature  and  concentration  of  the  substrate,  influence 
the  effects  of  the  reaction  of  the  medium.  Acids  act  as  zymo- 
exciters,  not  as  accessory  catalysors.  Other  substances  act  as 
zymoexciters :  the  chlorides  of  ammonium,  sodium,  and  potas- 
sium, though  not  to  marked  extent.  The  salts  of  the  heavy  met- 
als, cyanogen  compounds,  arsenic,  calcium  chloride,  and  alcohol 
act  as  negative  catalysors,  though  the  necessary  minimal  quan- 
tity varies  widely  with  different  preparations.  Chloroform  and 
toluol  in  moderate  quantity  are  of  no  effect.  Fluorescent  bodies, 
that  are  known  to  be  strong  oxydizing  agents  in  the  presence  of 
light  (Straub,  Edlefsen),  and  that  act  energetically  on  bacteria 
(Jodblauer  and  Tappeiner),  and  are  also  haemolytic  (Sacharoff 
and  Sachs),  are  active  depressors  to  the  action  of  invertase  (Em- 
merling),2  as  they  are  apparently  to  all  ferments,  as  well  as  to 
many  toxines.  A  solution  of  eosin  of  1 :1000000  will  depress  very 
materially  the  action  of  invertase.  Incidentally  remarked,  these 
are  the  only  substances  known  that  in  traces  depress  fermenta- 
tions like  the  so-called  poisons  of  reactions  described  by  Young, 
Silow,  and  others. 

Invertase  when  purified  to  the  highest  degree  yet  reported 
(Osborne,  probably  not  very  pure)  is  a  white  powder  that  is 
but  slightly  soluble  in  water,  forming  a  thick  viscid  suspension 
that  resists  filtration,  quite  in  contrast  to  the  behavior  of  amy- 
lase.  Unless  invertase  be  contaminated  with  some  vegetable  mu- 
cus or  gum,  it  is  certainly  the  most  colloidal  of  the  known  fer- 
ments. Nevertheless  the  active  principle  will  diffuse.  It  cannot 
be  gotten  ash-free.  It  contains  phosphorus  in  organic  combina- 
tion, and  on  being  boiled  with  acids  it  will  reduce  Fehling's  solu- 
tion, from  which  the  presence  of  a  carbohydrate  moiety  is  as- 
sured. It  does  not  give  the  biuret  test  and  is  very  resistant  to 
proteolysis— qualities  not  at  all  in  harmony  with  the  tentative 
classification  as  a  gly co-protein  that  would  be  suggested  by  its 
other  characteristics.  According  to  Hopner,  invertase  is  not  a 


VOL.  l]  Taylor — On  Fermentation.  189 

protein,  and  is  entirely  resistant  to  trypic  digestion.    For  maltase 
and  laetase  we  possess  still  less  data  upon  their  chemical  qualities. 

EMULSINE. 

The  fermentation  of  amygadeline  with  emulsine  was  described 
by  Robiquet  and  Boutron.  The  ferment  is  found  in  almonds,  in 
laurel  leaves,  in  many  plants,  in  a  large  number  of  fungi,  espe- 
cially the  parasitic  fungi  of  trees,  and  in  some  yeasts  like  as- 
pergillus  iiiger  and  penicillium  glaucum  (Herissey).  So  far  as 
known,  emulsine  is  not  found  in  the  animal  economy ;  the  alleged 
presence  in  the  digestive  juices  is  probably  due  to  the  presence 
of  bacteria,  many  of  which  produce  emulsine. 

Emulsine  is  able  to  ferment  a  large  number  of  glucosides.  and 
in  the  inversion  these  aether-like  bodies  are  split  into  d-glucose 
and  the  other  component. 

C13HU0T  +  H20  =  C6H1208  +  C7HS02. 
Salicin  +  water  =  d-glucose  +  saligenin. 

Emulsine  is  able  to  hydrolyze  salicine,  amygdaline,  arbutine, 
helecine,  asculine,  phloridzin,  coniferin,  gaultherin,  and  daphnin. 
As  a  matter  of  fact  emulsine  does  not  ferment  amygdaline  at 
all,  but  only  the  mandel-nitril  glucoside,  which  is  produced  by 
the  cleavage  of  the  amygdaline  under  the  influence  of  maltase. 
The  natural  glucosides  all  correspond  to  the  &-glucosides  (Pot- 
tevin).  The  number  of  glucosides  that  may  be  fermented  by 
this  one  ferment  has  always  attracted  attention,  and  these  facts 
have  been  quoted  as  illustrating  a  lack  of  ferment  specificity.  In 
truth,  the  inference  is  unwarranted.  We  are  dealing  writh  sugar 
fermentations :  these  cleavages  in  reality  represent  inversions, 
and  it  would  be  quite  natural  to  believe  that  it  is  the  sugar  moiety 
in  the  molecule  of  glucoside  that  determines  the  fermentability. 
Xow  in  all  the  glucosides  that  may  be  fermented  by  emulsine 
but  one  sugar  is  contained,  d-glucose.  Emulsine  is  not  able  to 
ferment  my  rosin,  xantorhammin,  rubian,  or  senalbine.  Two  of 
these  contain  no  d-glucose,  and  the  two  that  do  contain  it  contain 
not  one  but  two  other  molecules,  though  in  this  they  are  precisely 
comparable  to  amygdaline,  which  emulsine  does  not  ferment  di- 
rectly. It  must  not  be  understood  that  emulsine  is  stated  to  be 


190  I'/iift  rsity  of  California  Publications.      [PATHOLOGY 

able  to  split  all  such  glucosides  as  contain  d-glucose;  but  when 
all  the  glucosides  fermentable  by  emulsine  contain  this  hexose, 
the  behavior  of  this  ferment  may  at  least  not  be  urged  as  a  direct 
argument  against  the  specificity  of  its  action.  This  view  of  the 
matter  is  confirmed  by  the  observation  of  Fischer  that  an  in- 
version ferment  is  able  to  ferment  amygdaline;  the  pancreatic 
juice  is  also  known  to  be  able  to  ferment  amygdaline.  This  in- 
version ferment  is  in  all'  probability  maltase.  That  maltase  is  not 
able  to  ferment  other  natural  glucosides  might  be  due  to  the  fact 
that  they  contain  but  one  molecule  of  glucose  in  combination 
with  the  other  component,  while  amygdaline  contains  two  mole- 
cules, and  these  two  molecules  are  combined  in  a  manner  anal- 
ogous to  that  observed  in  maltose  and  in  the  o-methyl  maltosides 
and  glucosides.  Emulsine  for  its  part  is  also  able  to  accelerate 
the  hydrolysis  of  the  synthetic  methyl-glucosides,  but  only  those 
of  the  b  series.  This  all  is  in  harmony  with  the  fact  that  inver- 
tase,  so  far  as  known,  acts  only  on  those  disaccharides  as  contain 
d-laevulose,  and  ferments  also  only  those  synthetic  fructo-gluco- 
sides  of  the  a  series. 

Armstrong5  has  recently  attempted  to  define  the  point  of  at- 
tack in  the  hydrolysis  of  glucosides.  The  configuration  of  glu- 
cosides is  assumed  to  correspond  in  general  to  the  scheme : 


^ 

OH                        H 

Or* 

Ferment 

'H                 OH 

/ 

H                        OH                       OH 

•N                                                                        *»                                                                         TT 

GO*                     -                           g                       H- 

In  the  act  of  hydrolysis,  the  radicle  R  (the  aromatic,  aldehyde  or 
other  component  of  the  glucoside)  is  replaced  by  an  H;  in  the 
case  of  acids,  an  H  is  first  attached  to  the  adjacent  O,  while  in 
the  case  of  ferments,  the  attachment  may  occur  at  any  point 
along  the  chain  of  carbons.  Analogous  considerations  are  applied 
to  the  galactosides. 


VOL-!]  Taylor — On  Fermentation.  191 

Kinetics  of  the  fermentation.  Tammann  first  studied  the 
fermentation  of  emulsine  from  the  kinetic  point  of  view,  employ- 
ing salecin  and  amygdaline.  He  found  that  the  progress  of  the 
reaction  did  not  conform  well  to  the  regular  law  for  a  mono- 
molecular  reaction ;  the  march  was  slower  than  corresponded  to 
the  logary'thmic  curve.  In  general  terms  the  velocity  was  only 
roughly  proportional  to  the  concentration  of  the  substrate, 
though  for  concentrated  solutions  the  variations  were  wide.  The 
reaction  was  always  incomplete.  He  failed,  however,  to  bring 
about  a  direct  reversion  by  the  action  of  emulsine  upon  a  solu- 
tion of  the  products.  The  addition  of  products  depressed  the 
reaction,  and  the  entire  cessation  of  the  fermentation  could  be 
brought  about  by  the  early  addition  of  products.  The  addition 
of  substrate  to  a  quiescent  mixture  resulted  in  the  reestablish- 
ment  of  the  hydrolysis,  and  this  same  result  could  be  secured  by 
the  withdrawal  of  a  portion  of  the  products,  by  an  increase  in 
temperature,  by  dilution,  and  by  the  addition  of  additional  fer- 
ment. He  found  that  solutions  of  emulsine  undergo  hydrolysis, 
both  in  simple  solution  and  in  the  fermentation  test.  Tammann 
deserves  the  credit  of  having  first  approached  the  question  of 
the  kinetics  of  ferment  action  with  a  full  appreciation  of  the 
problem.  He  attempted  to  represent  the  progress  of  such  a  re- 
action in  an  equation  based  upon  the  idea  that  the  transformation 
was  in  the  unit  of  time  proportional  to  the  active  mass  of  the 
substrate,  and  of  the  non-inactivated  (or  better  undestroyed) 

ferment.  Thus  —  -^=C(A — x)  (F — y),  in  which  A  and  -Fare 
respectively  the  original  amounts  of  substrate  and  ferment,  x 
and  y  the  amounts  of  converted  substrate  and  destroyed  ferment 
in  the  time  t,  C  the  constant  of  velocity.  The  results  of  calcula- 
tions according  to  this  equation  do  not  give  good  results,  and  the 
reason  for  this  is  that  while  the  destruction  of  the  ferment  is  a 
simple  monomolecular  reaction  running  alongside  the  main  reac- 
tion, the  ferment  combines  with  the  components  of  the  main  re- 
action, and  during  these  combinations  it  is  protected  from  hy- 
drolysis. 

Henri0  has  lately  worked  over  the  action  of  emulsine  upon 
salacine.  He  confirmed  the  statements  of  Tammann  that  the 


192  University  of  California  Publications.      [PATHOLOGY 

reaction  proceeds  slower  than  represented  by  the  simple  loga- 
rythmal  curve,  that  the  proportionality  between  substrate  con- 
centration and  transformation  is  not  exact,  that  the  products 
depress  the  reaction,  and  that  the  velocity  was  in  a  general  way 
proportional  to  the  quantity  of  ferment.  He,  however,  found 
that  the  ferment,  in  diluted  systems  at  low  temperature  where 
the  fermentation  did  not  last  over  seven  hours,  was  preserved 
intact  for  all  practical  purposes.  He  then  attempted  to  apply  to 

1  A-\-x 

this   reaction   the   formula    C  =  —  log  .    -that    had   been   ob- 

I  A.  —  X 

tained  empirically  for  invertase,  but  since  the  reaction  is  slower 
than  corresponding  to  the  logarythmal  curve,  the  results  were 

worse  than  with  the  regular  equation  C  =  —  log  -7  Henri 

t  A.  —  X 

then  applied  his  theoretic  formula  as  described  under  invertase. 
The  two  applications  are  different  in  the  sense  that  applied  to 
invertase  m  (constant  of  equilibrium  with  substrate)  is  greater 
than  n  (constant  of  equilibrium  with  products),  while  in  the 
case  of  emulsine  n  is  greater  than  m,  corresponding  to  the  facts 
that  in  the  invertase  fermentation  the  progress  of  the  reaction  is 
more  rapid  and  in  the  case  of  the  emulsine  fermentation  slower 
than  the  logarythmal  curve.  He  ascertained  that  m  =  about  40, 
and  n  =  120.  When  the  experimental  data  was  calculated  ac- 
cording to  this  equation,  the  constants  were  fairly  uniform. 
While  Henri  ascribes  this  not  fully  exact  conformation  to  the 
not  entirely  accurate  figures  for  m  and  n,  one  cannot  resist  the 
feeling  that  this  may  have  been  due  to  inactivation  of  the  fer- 
ment. 

Visser  has  applied  his  equation  to  the  action  of  emulsine. 
For  this  ferment  he  has  determined  of  course  a  different  in- 
tensity of  action  than  that  of  invertase.  This  he  found  to  be  : 

1=       z__    —     —'t-    When  a  system  containing  a  gluco- 


side  and  emulsine  in  the  commonly  employed  concentration  is  at 
rest,  over  96  per  cent,  is  hydrolyzed  ;  therefore  the  intensity  formula 
may  be  inserted,  as  in  the  case  of  invertase,  into  the  simple  equation: 

This  gives  ™---= 


and  this  when  integrated  under  the  assumption  that  x—0  when  t= 
0,  yields:  2  CiC2  t  =8  A2  1    ,A_.-(-)A  +  (A-x))x.    The  results 


VOL.  1]  Taylor — On  Fermentation.  193 

are  not  satisfactory  in  that  the}-  do  not  yield  an  agreement  in 
the  constants  with  series  in  different  concentrations  of  the 
substrate. 

Herzog  has  also  applied  his  equation  to  the  reaction  of  the 
fermentation  of  glucosides.  The  results  were  fairly  concordant 
with  the  theory.  What  is  rather  surprising  in  the  results  of 
Henri,  Visser,  and  Herzog  is  the  concordance  in  the  results  de- 
spite the  fact  that  emulsine  is  one  of  the  most  sensitive  of  fer- 
ments. While  in  some  instances  it  is  true,  as  stated  by  Henri, 
that  the  ferment  will  preserve  itself  intact  through  a  test  of  not 
over  seven  hours,  this  is  very  liable  not  to  be  the  case;  many 
preparations  of  emulsine  will  not  yield  such  regular  results.  It 
is  quite  likely  that  the  intensity  (I)  of  the  equations  of  Henri  and 
Visser  include  the  factor  of  inactivation,  though  no  such  variable 
is  predicated  in  the  development  of  the  equations.  Probably  the 
inactivated  fraction  would  be  included  in  that  portion  supposed 
to  rest  in  combination  with  the  products. 

The  temperature  optimum  of  emulsine  lies  about  40-45°.  To 
a  certain  extent  it  varies  with  the  concentrations  in  the  system. 
Increases  in  temperature  are  accompanied  by  such  increase  in 
the  velocity  of  transformation  as  would  be  expected  from  the  law 
of  such  increase. 

The  reversion  of  the  glucosidic  fermentation  has  been  accom- 
plished by  Emmerling.  The  reaction  for  the  hydrolysis  of  amyg- 
daline  is  as  follows : 

(  ,,,ILT0HN+  2  H20  =  C0H5.COOH  +  HCN  +  2  C8H12O6. 
Amygdaline  +  water  =  benzaldehyde  +  HCN  +  d-glucose. 

This  reaction  has  never  been  reversed,  possibly  on  account  of 
the  action  of  the  hydrocyanic  acid  upon  ferments.  Fischer  has 
shown  that  a  ferment  exists  in  the  yeast  that  acts  to  split  amyg- 
daline  into  mandel-nitril  glucoside  and  d-glucose. 

QaH^OuN  +  H20  =  C14H1706N  +  C8H12O6. 

This  ferment  is  probably  maltase.  Emmerling  mixed  with  these 
substances  a  maltase  derived  from  yeast  and  months  afterwards 
isolated  amygdaline  from  the  mixture. 

Emulsine  is  not  a  resistant  ferment ;  it  may  be  easily  pre- 
served; it  is  quite  resistant  to  the  action  of  salts,  though  mod- 


194  University  of  California  Publications.      [PATHOLOGY 

erate  amounts  of  acid  or  alkali  depress  it.  The  optimum  reac- 
tion is  slightly  acid.  Emulsine  has  never  been  purified ;  it  is  not 
possible  to  separate  it  from  the  other  ferments  that  accompany 
it.  It  is  very  resistant  to  proteolytic  digestion.  The  ferment  is 
soluble  in  water  with  but  slight  opacity  and  without  marked  col- 
loidality;  it  may  be  filtered  through  infusorial  filters;  it  gives 
the  common  reactions  for  protein,  rotates  light  to  the  left,  and 
yields  on  heating  with  orcein  a  deep  orange  color  that  reminds 
one  of  pentose. 


FERMENTATION  OF  MONOSACCHARIDE.     ALCOHOLIC 
FERMENTATION. 

Of  the  numerous  fermentations  of  primary  sugars  those  af- 
fecting the  hexoses  are  best  known.  And  of  the  fermentations 
of  the  hexoses  that  affecting  d-glucose  and  leading  to  its  cleav- 
age into  aethyl  alcohol  and  carbon  dioxide  is  best  known,  and  to 
this  one  we  will  give  our  entire  attention.  The  scientific  study 
of  alcoholic  fermentation  dates  back  to  the  genial  prophet  of  the 
law  of  the  conservation  of  energy,  Lavoisier.  He  determined  that 
in  this  fermentation  the  sugar  is  converted  into  alcohol  and  car- 
bon dioxide,  both  bodies  containing  oxygen.  The  quantitative 
relations  were  determined  by  Gay-Lussac  and  Dumas,  who  wrote 
the  reaction  as  we  write  it  to-day.  It  is  very  interesting,  in  the 
light  of  modern  views  on  the  reversibility  of  ferment  action,  to 
observe  that  Lavoisier  was  convinced  that  were  it  possible  to 
combine  aethyl  alcohol  and  carbon  dioxide,  the  product  would  be 
glucose.  Neither  Lavoisier  nor  Gay-Lussac  paid  any  attention 
to  the  fungus  skum  that  is  a  macroscopic  appearance  in  all  alco- 
holic fermentations. 

Some  forty  years  later  de  Latour  and  Schwann  at  about  the 
same  time  independently  described  the  presence  of  yeast  cells  in 
fermentations,  and  stated  the  view  that  upon  the  life  processes 
of  these  vegetable  cells  depended  the  conversion  of  the  sugar  into 
alcohol  and  carbon  dioxide.  They  recognized  that  inoculation 
occurred  from  the  air,  and  considered  that  the  process  of  fer- 
mentation stood  in  some  relation  to  the  nutrition  of  the  plant. 


VOL.  1]  Taylor — On  Fermentation.  .       195 

They  indulged  in  little  speculation  upon  the  nature  of  the  phe- 
nomenon, and  though  'they  felt  the  truth,  they  did  not  extend 
their  studies  to  any  great  extent,  and  their  publications  had  little 
immediate  effect.  It  was  Pasteur  who  developed  this  theory, 
proved  its  truth  in  the  most  manifold  ways,  and  made  the  scien- 
tific and  industrial  world  believe  in  it.  Next  to  Pasteur  the 
biology  of  fermentation  owes  most  to  the  work  of  Hensen  and 
Duclaux. 

Prior  to  the  advent  of  Pasteur  upon  the  scene,  the  chemists 
simply  satirized  the  yeast  theory,  the  work  of  Pasteur  could  not 
be  thus  disposed  of,  and  was  bitterly  combatted.  Possibly  in  no 
scientific  discussion  has  the  personal  adjective  been  oftener  used. 
Even  now  the  latest  facts  in  the  general  theory  of  alcoholic  fer- 
mentation are  often  so  interpreted  and  expounded  as  to  consti- 
tute a  balm  to  the  historical  sense.  It  is  now  quite  the  fashion 
to  say  that  Liebig  and  Pasteur  were  both  in  the  right.  This  is 
not  true;  the  statement  does  no  credit  to  either  Pasteur  or  to 
Liebig,  both  of  whom  were  too  great  to  desire  spurious  reputa- 
tion, and  does  historical  injustice  to  another  man.  In  this  whole 
controversy  there  was,  upon  the  part  of  the  adherents  at  least, 
too  little  of  the  spirit  of  the  great  Faraday,  who  considered  a 
theory  as  a  question  put  to  Nature :  if  she  answered  yes,  well  and 
good ;  if  she  answered  no,  he  sought  another  path. 

Pasteur  demonstrated  that  in  fermentations  as  they  occur  in 
nature, -germs  are  always  present;  when  germs  are  excluded,  fer- 
mentation does  not  occur.  The  work  of  Schroeder  had  in  its 
technical  details  as  well  as  in  the%results  anticipated  many  of  the 
studies  of  Pasteur.  Mitscherlich  had  previously  shown  that  fer- 
mentation could  not  extend  through  a  diffusion  membrane,  and 
Helmholtz  had  confirmed  this  for  animal  membranes.  Years 
afterwards  Dumas  in  a  beautiful  experiment  showed  that  when 
two  layers  of  widely  different  specific  gravities  were  carefully 
placed  upon  each  other,  a  fermentation  would  not  pass  from  one 
layer  into  the  other.  Pasteur,  however,  went  beyond  the  demon- 
stration of  the  causative  relation  of  yeasts  to  fermentations,  as 
was  most  natural  he  developed  a  theory  of  fermentation.  This 
theory  was  in  brief  that  alcoholic  fermentation  was  the  result  of 
vegetation  without  air,  and  that  it  represented  a  compensatory 


196  University  of  California  Publications.      [PATHOLOGY 

functionation  whereby  the  yeasts  were  enabled  to  live  in  a  me- 
dium without  oxygen  by  making  the  oxygen  of  the  sugar  avail- 
able for  utilization.  It  was,  however,  promptly  shown,  especially 
by  Schuetzenberger,  that  alcoholic  fermentation  occurs  as  well 
in  the  open  atmosphere  and  even  in  an  atmosphere  of  oxygen  as 
under  anaerobic  conditions.  The  answer  of  the  Pasteur  party 
to  this  was  that  at  some  distant  past  time  the  yeast  had  acquired 
the  activity  as  a  so-to-speak  vicarious  function,  and  that  once  ac- 
quired the  function  continued  to  be  exercised  whether  the  condi- 
tions that  originally  called  it  into  being  were  present  or  not. 
This  application  of  the  doctrine  of  phylogeny  to  chemical  func- 
tion was  too  far  fetched  to  exert  a  prolonged  conviction.  Later 
in  his  life  Pasteur  modified  his  views  to  some  extent,  and  even 
attempted,  though  without  the  energy  born  of  conviction,  the 
isolation  of  a  ferment  from  the  bodies  of  the  yeast  cells ;  but  he 
never  yielded  the  general  idea  that  fermentation  constitutes  a  so- 
so-to-speak  vicarious  method  of  respiration, — every  yeast  cell  its 
own  oxygen  generator.  As  true  as  was  the  theory  of  Pasteur 
upon  the  biological  origin  of  natural  fermentations,  equally  im- 
probable was  his  theory  of  the  chemical  nature  of  that  fermenta- 
tion. Some  modern  botanists,  like  Stoklassa,  associate  the  func- 
tion of  alcoholic  fermentation  with  the  intracellular  respiration  of 
plants,  though  not  in  the  sense  employed  by  Pasteur. 

The  theory  of  Liebig  sounds  to  us  to-day  strangely  mystical. 
According  to  him,  the  fermentation  of  a  sugar  was  a  process 
which  the  sugar  contracted,  so  to  speak,  from  contact  with  some 
other  body  undergoing  the  same  or  some  similar  disintegration. 
The  cause  of  fermentation,  he  thought,  lay  in  the  tendency  pos- 
sessed by  substances  in  process  of  chemical  action  to  convey  this 
same  chemical  action  to  another  body  in  proximity  to  it,  or  at 
'  least  to  make  the  neighboring  body  susceptible  to  it.  The  nature 
of  the  transmission  he  conceived  to  be  a  sort  of  molecular  vibra- 
tion. When  a  body  in  process  of  decomposition  lay  beside  an- 
other body,  the  molecular  vibrations  were  transferred  to  the 
second  body  with  the  result  that  it  thereupon  underwent  the 
same  decomposition.  Ferments  were  thus  defined  as  bodies  in 
process  of  disintegration,  and  the  alcoholic  fermentation  was  the 
result  of  the  disintegration  communicated  to  the  sugar  molecule. 


VOL.  l]  Taylor — On  Fermentation.  197 

The  conception  was  applied  to  all  the  animal  ferments  by  Naegeli. 
When  Liebig  finally  realized  the  force  of  the  experiments  of 
Pasteur  that  fermentations  never  occur  in  the  absence  of  germs, 
he  shifted  his  ground  by  locating  the  decomposing  body  in  the 
yeast  cell.  It  is  an  error  to  state  that  Liebig  simply  fought  for 
a  chemical  interpretation  of  fermentation  as  against  a  vitalistic 
interpretation.  Liebig  contended  as  well  for  a  particular  chem- 
ical interpretation  of  fermentation,  after  as  much  as  before  his 
admission  of  the  etiological  relations  of  the  yeast  germ. 

Very  recently  Barendrecht  has  proposed  a  hypothesis  for  fer- 
mentation that  recalls  vividly  the  hypothesis  of  Liebig.  Baren- 
drecht considers  the  catalytic  activity  of  a  ferment  to  lie  in 
emanations  that  proceed  from  it  and  act  upon  surrounding  mole- 
cules. This  idea  is  obviously  a  far-fetched  application  of  the 
earlier,  now  admittedly  erroneous  interpretation  of  the  phenom- 
enon of  so-called  induced  radio-activity.  The  emanations  are 
considered  to  be  absorbed  by  the  substrate  and  the  products  of  a 
reaction,  and  upon  this  an  equation  is  developed.  Now  it  cannot 
of  course  be  denied  that  the  acceleration  of  a  reaction  through 
the  medium  of  intermediary  reactions  may  be  due  to  the  action 
of  emanations,  electronic  or  otherwise,  but  it  is  the  wildest  specu- 
lation. The  sympathetic  reference  to  the  n-rays  indicates  clearly 
that  the  author  is  emanationally  inclined.  For  us  it  is,  in  the 
present  state  of  our  knowledge,  sufficient  to  dismiss  the  subject 
with  the  remark  that  the  sheep  of  Badenrecht  is  only  the  wolf  of 
Liebig4  dressed  up  in  modern  emanation  wool. 

At  the  very  height  of  this  controversy,  1858,  what  we  to- 
day regard  as  the  true  theory  of  fermentation  was  enunciated 
by  Moritz  Traube.  It  was  not  a  casual  statement,  but  a  per- 
fectly conscious  and  deliberated  promulgation  of  a  definite  the- 
ory. Traube  was  a  resourceful  theoretical  as  well  as  a  brilliant 
qualitative  experimental  chemist.  He  had  worked  much  upon 
the  qualitative  nature  of  reactions,  and  especially  upon  catalytic 
reactions.  He  had  a  fine  sense  of  perspective,  and  was  content 
to  state  his  theory  without  becoming  engaged  in  the  contempo- 
raneous polemic  and  to  await  the  future  experimental  develop- 
ment of  that  theory.  He  formulated  the  view  that  there  was  in 
the  body  of  the  yeast  cell  a  product  of  its  metabolism,  though  in 


198  r  Diversity  of  California  Publications.      [PATHOLOGY 

no  concrete  way  defined,  a  substance  which  once  existent  reacted 
with  sugar  with  the  production  of  alcohol  and  carbon  dioxide. 
And  secondly  this  reaction  between  the  hypothetical  substance 
and  the  sugar  was  a  reaction  entirely  independent  of  the  parent 
cell,  and  in  its  chemical  nature  and  relations  similar  and  related 
to  the  catalytic  reactions.  Traube  knew  that  the  catalytic  de- 
compositions occurred  slowly  in  the  absence  of  the  catalysor. 
The  definite  separation  of  the  reaction  de  novo  and  the  accel- 
erated reaction  is,  however,  due  largely  to  Ostwald,  who  has 
through  his  work  secured  the  general  application  in  chemical 
reasoning  of  this  distinction.  The  work  of  the  last  forty  years 
has  resulted  in  the  verification  of  the  theory  of  Traube,  though 
he  did  not  live  to  witness  the  isolation  of  zymase.  The  work  of 
Traube  was  quickly  recognized  by  Berthelot  and  Claude  Bernard 
in  France,  and  by  Scloenbein,  Hoppe-Seyler,  and  von  Manassein 
in  Germany.  Bernard  attempted  the  isolation  of  the  ferment,  as 
did  Naegeli,  Loew,  Mayer,  von  Manassein,  and  even  Pasteur,  all 
with  negative  results.  In  1897  E.  Buchner1  isolated  the  active 
substance  in  a  cell-free  form.  Miquel  had  previously  isolated  the 
ferment  urase  from  the  micrococcus  urea. 

When  we  speak  of  the  isolation  of  the  alcoholic  ferment  from 
the  yeast  cells,  we  mean  as  yet  only  isolation  in  the  same  rough 
sense  that  the  term  was  used  in  connection  with  amylase,  and 
invertase.  What  is  obtained  is  a  fluid  extract  of  the  yeast  cells, 
and  this  contains  among  other  inorganic  and  organic  constituents 
the  active  ferment.  This  extract  has  been  purified  to  some  ex- 
tent, but  up  to  the  present  zymase  has  not  been  prepared  in  as 
pure  condition  as  many  of  the  other  ferments.  The  method  of 
preparation  of  the  extract  is  simple.  The  yeast  (zymase  is  found 
in  many  fungi)  is  first  washed  until  all  the  particles  of  light 
gravity  have  been  removed  and  the  wash  water  flows  clear ;  this 
is  best  done  by  a  combination  of  straining  and  decantation.  It 
is  then  dried  under  pressure,  about  fifty  atmospheres  being  neces- 
sary. The  resultant  powder  is  still  two-thirds  water.  This  pow- 
der is  then  mixed  with  an  equal  weight  of  quartz  sand  of  finest 
grain  and  about  one-fourth  weight  of  infusorial  earth  that  has 
been  washed,  ground,  and  calcined.  This  mixture  is  then  ground 
to  a  fine  powder,  as  determined  by  microscopic  appearances. 


VOL.  l]  Taylor — On  Fermentation.  199 

Macroscopically  the  powder  assumes  a  plastic  dough-like  consist- 
ency, due  to  the  absorption  of  the  cellular  juices  by  the  infusorial 
earth.  When  the  microscopic  examination  indicates  that  nearly 
all  the  cells  have  been  crushed,  the  mass  is  placed  in  a  heavy 
canvas  cloth  and  submitted  in  a  hydraulic  press  to  a  pressure  of 
some  300  atmospheres.  In  all  probability  the  new  method  of 
MacFadyean  and  Rowland  for  grinding  germs  at  the  tempera- 
ture of  liquid  air  would  yield  better  results.  Not  only  is  the 
trituration  of  the  cells  accomplished  best  at  this  low  temperature 
on  account  of  the  increased  brittleness  of  the  tissue,  but  the  mass 
would  not  assume  such  a  plastic  consistency  on  account  of  the 
freezing  of  the  water;  disintegration  of  the  juice  also  would  be 
inhibited.  The  yield  of  juice  is  about  three-fourths  of  the  water 
content  of  the  yeast;  a  second  extraction  after  regrinding  will 
yield  a  little  more.  The  juice  should  be  filtered,  and  kept  at  very 
low  temperature. 

A  more  recent  method  developed  in  the  laboratory  of  the 
pharmacologist,  H.  H.  Meyer,  yields  much  better  results.  The 
yeast  cells  are  simply  placed  in  a  vacuum  and  the  space  filled 
with  ether  vapor.  Fluid  soon  begins  to  pass  from  the  cells;  this 
fluid  is  rich  in  zymase,  and  may  be  recovered  by  simple  filtration 
with  pressure.  Just  how  the  procedure  operates  is  not  clear.  As 
Loeb  has  suggested,  one  might  infer  the  process  to  be  autolytic. 
^Yhat  one  cannot  in  addition  understand  is  the  process  of  diffu- 
sion, since  it  has  been  shown  that  in  natural  alcoholic  fermenta- 
tion the  reaction  occurs  within  the  cells,  the  sugar  diffuses  in  and 
the  products  out,  but  the  ferment  does  not  and  apparently  can- 
not diffuse  out. 

The  yeast  juice  is  of  a  yellowish  color,  somewhat  opalescent 
(due  in  part  to  colloidal  infusorial  earth)  ;  a  slightly  viscid  fluid, 
practically  neutral,  with  the  odor  and  taste  of  yeast.  After  fil- 
tration through  an  infusorial  filter,  the  fluid  is  as  clear  as  water, 
and  does  not  display  more  than  a  trace  of  opacity  when  illumi- 
nated with  oblique  light.  Filtration  through  an  infusorial  filter 
usually  carries  with  it  loss  in  enzymic  power,  though  it  varies 
much  with  different  preparations  and  is  least  when  done  at  the 
lowest  feasible  temperature.  The  loss  during  such  a  filtration 
is  highest  at  the  beginning;  if  one  collects  only  the  last  half  of 


200  University  of  California  Publications.      [PATHOLOGY 

a  large  filtration,  the  activity  will  be  found  not  seriously  reduced. 
Porcelain  filters  work  more  slowly,  and  do  not  seem  to  give  better 
results.  Such  filtration  is  necessary  to  exclude  isolated  yeast 
cells,  bacteria,  and  also  the  colloidal  infusorial  earth. 

The  yeast  juice  will  not  dyalize  through  a  parchment  mem- 
brane with  measurable  rapidity.  This  observation  is  in  accord 
with  the  experience  that  it  has  never  been  possible  to  detect  fer- 
mentative action  in  the  fluid  in  which  yeast  cells  are  suspended. 
Groblewski,  for  example,  filtered  through  a  sand  filter  a  solution 
of  sugar  containing  yeast  and  presenting  active  fermentation, 
but  the  filtrate  was  entirely  inactive.  This  practical  inability  of 
the  ferment  to  dyalize  is  interpreted  to  indicate  that  in  nature 
the  fermentation  occurs  within  the  cell,  the  sugar  dyalizing  in 
and  the  alcohol  and  carbon  dioxide  diffusing  outwards. 

The  extract  is  stated  not  to  polarize  light.  It  has  a  specific 
gravity  of  1030  to  1050,  contains  from  11  to  14  per  cent,  of  solids, 
from  1.5  to  2  per  cent  of  ash,  one-fifth  per  cent,  of  phosphorus, 
and  from  13  to  15  per  cent,  of  nitrogen ;  that  is,  the  solids  con- 
tain some  13  to  16  per  cent  of  nitrogen.  The  extract  is  very  rich 
in  protein,  gives  the  biuret,  Millon,  and  Xanthoproteic  reactions, 
and  contains  more  or  less  glycogen.  The  proteins  have  a  low 
coagulation  point;  even  at  45°  the  first  coagulation  is  noted, 
while  complete  coagulation  occurs  at  a  little  over  60°.  The  addi- 
tion of  strong  acids  or  alkali  will  precipitate  heavily  in  the  cold. 
As  indicated  by  the  rather  high  content  in  phosphorus,  the  pro- 
tein consists  largely  of  neucleo-proteid,  and  this  is  confirmed  by 
the  appearance  of  purin  bases  on  digestion.  On  autodigestion 
the  usual  amido-acids  noted  in  protein  digestions  in  general  are 
produced,  and  also  purin  bases. 

The  yeast  extract  possesses  rather  marked  powers  of  reduc- 
tion. Thus  nitrites  are  reduced  with  the  production  of  gaseous 
nitrogen ;  hyposulphites  are  reduced  to  hydrogen  sulphide ;  alka- 
line copper  and  silver  solutions  are  promptly  reduced  in  the 
cold;  sulphur  is  converted  into  H2S,  and  also  into  mercapten. 
These  reductions  are  not  dependent  upon  the  zymase;  they  de- 
pend upon  bodies  that  are  soluble  in  alcohol,  and  remain  after 
the  ferment  is  destroyed.  The  ability  to  reduce  methylen  blue, 
however,  disappears  with  the  destruction  of  the  ferment. 


VOL.  l]  Taylor — On  Fermentation.  201 

The  extract  is  very  unstable  from  the  standpoint  of  the 
zymase.  This  liability  seems  to  be  due  to  autodigestion ;  that  is, 
to  the  digestion  of  the  specific  ferment  by  the  active  proteolytic 
ferment  present  in  the  extract.  A  low  temperature  retards  this 
digestion.  The  destruction  of  the  zymase  in  the  extracts  Buchner 
found  entirely  independent  of  atmospheric  oxygen.  That  the 
digestion  is  the  cause  of  the  destruction  of  the  zymase  is  ren- 
dered very  probable  by  the  fact  that  the  destruction  of  zymase 
and  the  diminution  in  the  coagulable  protein  diminish  pari 
passu.  "When  dessicated  at  low  temperature  in  vacuo  the  powder 
is  very  stable,  can  be  conserved  a  long  time  without  serious  loss 
of  specific  activity,  and  bears  heating  up  to  100°. 

This  extract  contains  a  number  of  ferments:  a.  the  alcoholic 
ferment,  zymase;  6.  the  proteolytic  ferment,  yeast-endotryptase 
a  very  active  and  destructive  ferment;  c.  invertase;  d.  maltase; 
e.  some  form  of  amylase  since  glycogen  is  hydrolyzed,  and  even 
starch,  though  very  slowly ;  /.  substances  that  will  react  with  hy- 
drogen peroxide,  and  the  reducing  bodies  already  mentioned, 
though  what  relations  these  may  bear  to  the  enumerated  fer- 
ments are  not  known.  Of  the  sixteen  isomeric  hexoses  (twelve  of 
which  have  been  prepared)  the  alcoholic  ferment  in  zymase  is 
able  to  accelerate  the  fermentation  of  but  four:  d-glucose,  d- 
mannose,  d-fructose,  and  d-galactose,  the  last  with  difficulty. 
The  extract  may  be  concentrated  by  careful  freezing;  the  last 
fluid  is  very  rich  in  ferments.  Many  attempts  have  been  made 
to  free  the  zymase  from  the  other  bodies.  The  least  unsuccessful 
methods  have  been  by  precipitation  with  alcohol-aether  and  with 
acetone.  Through  these  procedures  some  of  the  alcoholic  ferment 
is  lost,  and  much  of  the  other  material  retained.  The  precipi- 
tated powders  keep  well,  but  it  is  doubtful  if  the  procedures  are 
of  much  value  from  any  other  point  of  view.  The  multiplicity 
of  ferments  in  the  extract  of  one  yeast  illustrates  how  worthless 
have  been  the  discussions  as  to  whether  the  fermentative  activ- 
ities in  crude  amylases,  invertases,  etc.,  were  due  to  one  or  more 
ferments,  and  whether  all  the  invertases  were  alike,  etc. 

The  yeast  extract  will  invert  sacchrose  and  maltose  rapidly, 
and  will  ferment  alcoholically  d-glucose  and  d-laevulose  with 
rapidity.  Raffinose  is  slowly  fermented,  glycogen  and  d-galac- 


202  University  of  California  Publications.      [PATHOLOGY 

tose  very  slowly,  while  starch  is  hydrolyzed  with  the  greatest 
difficulty.  Lactose  is  not  inverted  at  all.  These  activities  corre- 
spond in  general  with  those  noted  for  fermentation  with  yeast, 
except  that  yeast  cannot  ferment  glycogen  at  all ;  this  is  supposed 
to  be  due  to  inability  of  diffusion  upon  the  part  of  the  glycogen. 

Zymase  appears  to  act  best  in  a  reaction  of  faint  alkalinity. 
If  the  fermentation  be  done  in  a  closed  chamber,  it  is  apparent 
that  when  the  fluid  becomes  saturated  with  carbon  dioxide  no 
alkaline  reaction  can  be  maintained,  and  the  conditions  would  be 
as  they  are  in  the  blood,  practical  neutrality.  All  acids  seem  to 
depress ;  partly  by  accelerating  the  digestion  by  the  endotryptase, 
and  partly  by  true  inactivation.  Neutral  salts  possess  either  no 
action,  or  they  depress  the  fermentation.  Chloroform,  toluol, 
thymol,  and  glycerine  have  little  action.  Weak  solutions  of  arse- 
nite  do  not  inhibit ;  strong  solutions  do  so,  as  do  all  cyanogen  com- 
pounds. On  account  of  the  complicated  conditions,  referable 
especially  to  the  endotryptase,  the  data  upon  the  influence  of  ex- 
traneous substances  are  difficult  to  value.  The  alkali  phosphates 
(the  P04  is  the  active  factor)  are  group  zymo-exciters  to  zymase, 
as  is  curiously  enough  also,  lecethin.  When  yeast  is  allowed  to 
lie  for  some  time  in  a  solution  of  cane  sugar  containing  aspara.uin, 
the  zymase  from  it  will  be  found  very  active.  (Buchner.) 

Harden1  has  investigated  in  detail  the  zymoexcitor  for  alco- 
holic fermentation.  While  not  denying  the  activity  of  phos- 
phates, he  points  out  that  an  organic  substance  in  the  yeast 
extract  is  much  more  potent.  If  a  yeast  extract  be  allowed  to 
undergo  autodigestion,  then  boiled  and  filtered,  this  fluid  is  most 
active  as  a  zymoexciter.  The  active  substance  may  be  precipi- 
tated by  alcohol  at  75  per  cent,  concentration,  is  thermo-stabile 
in  solution,  but  is  destroyed  on  being  ashed.  The  substance  dif- 
fuses easily;  it  is  therefore  crystalloid.  Harden  goes  so  far  as  to 
suggest  that  zymase  alone  cannot  accelerate  the  reaction  of  fer- 
mentation, but  the  experimental  evidence  is  not  sufficient  to  sup- 
port this.  Certain  it  is,  however,  that  the  zymoexciter  exerts  a 
most  marked  stimulation  to  the  fermentative  action  of  the  zymase. 
The  zymoexciter  alone  is  not  an  alcoholic  ferment. 

Buchner  and  Albertoni  believe  that  the  substance  in  yeast  ex- 
tract described  by  Harden  and  Young  as  a  co-ferment  for  zymase 


VOL.  1]  Taylor — On  Fermentation.  203 

is  phosphoric  acid.  This  accelerates  markedly  the  fermentation 
by  zymase.  Organically  combined  acid,  as  in  lecethin,  had  the 
same  action. 

Harden  and  Young  have  shown  that  when  a  phosphate  is 
added  to  a  fermenting  system,  it  is  on  the  completion  of  the  reac- 
tion not  recoverable  by  precipitation  with  magnesia  mixture  or 
silver  nitrate. 

Stoklasa  has  recently  published  the  statement,  accompanied 
by  the  experimental  data,  that  he  has  been  able  to  isolate  zymase 
from  different  plants;  and  also  from  mammalian  tissues  a  cell- 
free  extract  that  has  the  power  of  fermenting  sugar.  The  ex- 
periments are  convincing  (assuming  that  the  bacteria  have  been 
effectively  excluded,  which  has  been  denied  by  Maze  and  others), 
but  require  confirmation.  Should  the  observation  prove  to  be 
correct,  the  fact  will  constitute  one  of  fundamental  physiological 
importance,  since  the  modus  of  sugar  combustion  will  be  thereby 
elucidated.  The  presence  of  alcohol  in  mammalian  tissues  was 
long  ago  determined  by  Hoppe-Seyler  and  Akari,  Rajewski  and 
Bechamp.  Maignon  has  recently  gone  over  the  ground  anew,  and 
found  alcohol  invariably  in  muscle,  even  after  death. 

The  reaction  of  alcoholic  fermentation.  The  auto-fermenta- 
tion of  d-glucose  has  been  demonstrated  by  Duclaux.  He  ex- 
posed sterile  solutions  of  the  sugar,  of  a  faint  alkaline  reaction, 
to  sunlight,  and  after  a  time  wras  able  to  demonstrate  the  pres- 
ence of  aethyl  alcohol.  Colloidal  platinum  acts  also  as  an  accel- 
erator to  the  auto-reaction.  The  simple  reaction  is  C6H1206  = 
2  C2H60  -)-  2  C02.  The  equation  obviously  represents  but  the 
initial  and  the  final  stages.  It  is  not  possible  to  write  a  rational 
equation  based  upon  the  sterio-isomeric  formula  of  d-glucose  that 
will  illustrate  the  direct  cleavage  of  the  sugar  into  alcohol  and 
carbon  dioxide.  Intermediary  reactions  must  be  assumed,  and 
in  all  probability  these  demand  the  temporary  addition  of  other 
elements.  Water  is  quite  certainly  engaged  in  the  reaction  of 
fermentation.  Baeyer  proposed  the  first  explanation  as  early  as 
1870.  His  conception  was  that  water  was  added  to  the  molecule 
of  sugar  in  what  we  would  now  call  ionic  form;  following  this 
the  hydroxyl  groups  underwent  a  rearrangement  whereby  they 
were  accumulated  at  two  points,  with  the  consequent  reduction 


204  University  of  California  Publications.      [PATHOLOGY 

of  two  other  groups;  the  carbon  chain  was  then  broken  at  the 
points  of  the  accumulation  of  oxygen.  The  theory  holds  for 
simpler  compounds  as  well,  and  the  processes  are  quite  identical 
with  those  frequently  observed  in  connection  with  the  action  of 
those  substances  that  at  high  temperatures  effect  the  withdrawal 
of  water  from  an  organic  molecule. 

When  propyl  alcohol  is  heated  with  sulphuric  acid  it  is  trans- 
formed into  propylen,  which  in  its  turn  again  binds  water  in  a 
different  manner  to  form  isolpropyl  alcohol. 

Propyl  alcohol  minus  water  =  propylen. 
CH3.CH2.CH2OH  —  H20  =  CH3.CH.CH2. 
Propylen  plus  water  =  isopropyl  alcohol. 
CH3.CH.CH,.  +  H2Or=CH3.CH(OH).CH3. 

The  process  consists  obviously  in  the  transfer  of  one  hydroxyl 
group  from  one  atom  of  carbon  to  another.  Another  frequent 
illustration  of  such  a  transfer  of  hydroxyl  groups  from  one  car- 
bon to  another  is  seen  in  the  rule  that  in  the  use  of  substances 
that  abstract  water,  the  group  CH(OH).CH(OH).  passes  into 
CH2.CO,  the  reaction  passing  through  the  intermediary  unsat- 
urated  alcohol  CH:C(OH)  :.  In  a  similar  manner  oxalic  acid  is 
split  into  formic  acid  and  carbon  dioxide,  etc.  That  high  tem- 
perature is  not  necessary  has  been  shown  by  Wohl  and  Oesterlin, 
who  have  shown  that  d-tartaric  acid  (COOH.CH(OH).CH(OH). 
COOH.)  through  the  successive  actions  of  aectyl  chloride  and 
pyridine  may  be  converted  at  low  temperature  into  oxalacetic 
acid  (COOH.CO.CH2.COOH).  The  reactions  whereby  hexoses 
may  be  converted  into  each  other  by  the  action  of  alkali  has  also 
been  explained  in  an  analogous  manner  by  Lobry  de  Brun  and 
Van  Erenstein. 

The  formation  of  acroline  from  glycerine  is  represented  by 
the  following  series : 

Glycerine  Acroline 

CH2.OH1  fCH2         1  fCH2  fCH2 

I     minus     I  !     plus  minus   |  || 

CH  .OH  }-2H20=-j  C  ^H,0=^CH  H,O=  -j  CH 


CH2.OHJ  LCH.OH  j  Lcn.(OH).2  [CH.O 

Applied  to  the  fermentation  of  d-glucose  we  have  the  following 
stages. 


VOL.  1] 


Taylor — 0 n  Fermentation. 
•  \  «,<- 


205 


:6H12O6  +  4  H2O 

Pt  _j_  2  H20                  P.,  +  3  H2O               End-products 

:'H.,OH    H  H 

:HOH 
:HOH    OH  OH 

CH3                 (J  M 
CHOH 
OH 
C                      OH 
OH 

CH3 
JHOH           H 
OH 

C             OH    H 
\ 
OH 

CH3 
CH2OH 

CO2  +  2  H2O 

— 

\ 

O 

OH 

OH 

3HOH     OH  OH 

1 
DHOH 

OHO         H  H 

C                     OH 
OH 
CHOH 
CH2OH          HH 

C             OH    OH 
\ 
OH 

CHOH         H 
CH3 

CO2  +  2  H2O 

CH2OH 
CH3 

This  equation  illustrates  that  it  is  possible  to  write  the  reaction 
for  the  fermentation  of  d-glucose  with  but  the  single  assumption 
of  the  successive  addition  and  subtraction  of  water,  with  the 
translocation  of  hydroxyl  groups.  The  last  intermediary  stage  i 
represents  the  anhydride  of  lactic  acid.  The  reaction,  i.e.,  the 
production  of  alcohol,  has  never  been  accomplished  for  d-glucose 
by  the  simple  action  of  heat  alone ;  thus  far  the  yield  has  been 
lactic  acid  and  not  aethyl  alcohol.  Quite  similar  conceptions 
have  been  advanced  by  Rayman,  Mayer,  Wagner,  and  by  Wohl. 
Harden'2  has  recently  studied  the  fermentation  of  d-glucose  by 
the  Bac.  coli  communis.  He  found  that  alcohol,  lactic  and  acetic 
acids  were  regularly  produced,  and  believed  that  the  acids  are 
produced  from  the  sugar  directly  and  not  from  the  alcohol  by 
oxidative  fermentation.  He  found  that  the  yield  in  alcohol  was 
least  in  the  fermentation  of  d-glucose,  greater  in  mannite,  and 
greatest  of  all  in  glycerine.  This  fact  he  brought  in  relation  to 
the  number  of  CH2OH.CHOH  groups,  and  postulated  the  view 
that  the  aethyl  alcohol  was  derived  only  from  this  group.  Thus 
for  glycerine  we  have 

CH2OH  CH3.CH2OH       aethyl  alcohol 


CHOH 
CH,OH 


H.COOH. 


formic  acid 


206 


University  of  California  Publications.      [PATHOLOGY 


Mannite  yields  for  each  molecule  one  of  aethyl  alcohol,  as  is  seen 
in  the  equation,  written  for  two  molecules. 


Mannite. 
CH2OH 

CHOH  CH2OH 

CHOH  CHOH 

CHOH  CHOH 

CHOH  CHOH 

CH2OH  CHOH 


=  CH3.CH2OH.  +  C02  +  H2 


=  lactic  acid,  ect. 


=  CH3.CH2OH.  +  CO2  +  H2 


In  the  case  of  d-glucose,  on  the  contrary,  one  molecule  of  aethyl 
alcohol  is  produced  for  each  two  molecules  of  sugar. 


d-glueose. 
CH2OH 

CHOH       CH,OH 


CHOH  CHOH 
CHOH  CHOH 
CHOH  CHOH 


=  CH3.CH2OH.  +  CO2 


=  lactic  acid,  ect. 


CHO  CHOH 

CHO        +  H20  =  CH3.COOH  +  CO,  +  H2 

Now  this  closely  resembles  the  Baeyer  scheme.  Granted  for  the 
sake  of  argument  that  the  equation  represents  a  single  process, 
it  is  apparent  that  it  will  not  apply  to  the  alcoholic  fermentation 
of  d-glucose  by  zymase  because  the  entire  mass  of  sugar  appears 
as  alcohol  and  carbon  dioxide.  Under  such  circumstances  alcohol 
must  come  from  the  numerous  CHOH  groups,  and  the  Baeyer 
theory  represents  the  simplest  conception  of  securing  aethyl  alco- 
hol from  these  groupings.  Indeed  it  is  apparent  that  the  inter- 
mediary reactions  in  the  Baeyer  equation  can  be  written  almost 
directly  into  the  Harden  equation. 

This  theory  of  course  does  not  explain  the  reaction  of  fer- 
mentation in  the  desired  concrete  sense.  But  it  does  place  the 
reaction  upon  the  same  plane  as  many  other  reactions  of  organic 


VOL.  1] 


Taylor — On  Fermentation. 


compounds,  and  thus  the  problem  of  alcoholic  fermentation  be- 
comes simply  one  of  the  problems  connected  with  reactions  in 
the  asymetrical  carbon  compounds.  That  the  Baeyer  theory  fits 
well  into  the  doctrine  of  catalytic  acceleration  will  become  ob- 
vious when  we  consider  the  catalyses  from  the  point  of  view  of 
successive  intermediary  reactions. 

From  the  standpoint  of  organic  chemical  behavior  the  Baeyer 
theory  has  one  doubtful  step,  the  reduction  of  the  aldehyde 
group.  Buchner  and  Meisenheimer  have  recently  worked  upon 
the  matter  experimentally,  and  have  developed  a  different  for- 
mulation of  the  equation  that  not  only  avoids  the  supposition  of 
a  reduction  of  the  aldehyde  group,  but  also  indicates  the  forma- 
tion of  alcohol  from  the  CHOH  group. 


CHO 

CHOH 
I 
CHOH 

CHOH 


HoO. 
OH 
OH 

II 

H 
OH 

OH 
H 
H 

= 

Pi  +  H20. 

COOH 

1 
CHOH 

CHo            H 

1 
1 
CO           OH 

1 

CHOH 

1 

CH2 

= 

Lactic-acid.   2  H2O. 
COOH             OH 
CHOH            H 
CH3. 

= 

Products 
CO, 
CH2.OH 
CH3. 

COOH          OH 
CHOH         H 
CH3. 

CO2 
CH2.OH 
CH3. 

CH,OH 


They  were  able  to  detect  the  transient  presence  of  lactic  acid 
in  the  system,  and  they  have  therefore  revived  the  older  theory 
that  possibly  two  ferments  might  be  concerned,  one  transforming 
the  sugar  into  lactic  acid,  the  other  converting  the  lactic  acid 
into  alcohol  and  carbon  dioxide.  For  this  suggestion  they  were 
able  to  produce  no  experimental  evidence,  and  there  can  be  no 
doubt  that  the  general  tendency  expressed  in  such  a  suggestion 
is  unsound.  If  we  are  to  assume  a  different  ferment  or  catalysor 
for  each  successive  intermediary  stage  in  the  reactions  concerned, 
we  shall  soon  be  in  the  darkness  of  hopeless  confusion.  The  ten- 
dency to  invoke  new  powers  when  difficulties  present  themselves 
is  one  unfortunately  deeply  grounded  in  biological  studies  with 
chemical  and  physical  aspects.  When  the  data  are  not  sufficient 
to  permit  us  to  decide  a  question  upon  the  scope  of  the  known 


208  University  of  California  Publications.      [PATHOLOGY 

factors,  that  same  data  cannot  be  employed  to  justify  the  invo- 
cation of  new  factors.  The  mere  fact  that  we  cannot  understand 
how  one  ferment  could  ferment  d-glucose  to  lactic  acid  and  also 
ferment  lactic  acid  to  alcohol  and  carbon  dioxide  is  no  reason  in 
our  present  state  of  knowledge  for  inferring  that  two  ferments 
must  be  concerned.  It  cannot,  on  the  other  hand,  be  denied  that 
two  ferments  might  be  concerned. 

In  a  more  recent  communication  Buchner  and  Meisenheimer 
infer  methyl  glyoxal  to  be  the  intermediary  stage  between  sugar 
and  lactic  acid.  They  were  able  to  obtain  lactic  acid  as  well  as 
alcohol  as  the  result  of  the  action  of  alkali  on  sugar.  They  are 
definitely  convinced  of  the  plurality  of  the  zymase,  and  speak  of 
zymase  and  lactacidase.  Stoklasa  in  a  recent  paper  gives  expres- 
sion to  the  same  assumption  of  the  dual  nature  of  zymase.  The 
zymase  is  held  to  act  to  the  stage  of  lactic  acid,  the  lactacidase 
following  this  to  the  stage  of  alcohol. 

Another  scheme  for  the  reactions  has  been  given  by  Erlen- 
meyer.     CH2OH  —  CHOH  —  CHOH  —  CHOH  —  CHOH  — CH 
0.  This  undergoes  intramolecular  rearrangement  into  CH3  —  CO 
-  CHOH  —  CH2  —  CO  —  CHO.     This  is  an  aldo  condensation 
product  of  two  molecules  of  pyro-tartaric  aldehyde,  CH3  —  CO 
—  CHO.     This  substance  passes  easily  into  lactic  acid,  CH3  — 
CHOH  —  COOH  by  the  addition  of  water.     The  lactic  acid  is 
then  converted  into  alcohol  as  in  the  other  schemes. 

The  experiences  with  zymase  have  thrown  a  very  interesting 
light  upon  the  qualitative  reaction  in  an  alcoholic  fermentation. 
From  the  earliest  days,  indeed  since  very  shortly  after  Gay- 
Lussac  studied  the  reaction,  the  fermentation  of  a  primary  sugar 
has  been  supposed  to  yield  four  products — aethyl  alcohol,  carbon 
dioxide,  succinic  acid,  and  glycerine.  When  it  came  to  the  con- 
sideration of  the  quantitative  yield,  the  complexity  became  ap- 
parent. In  the  attempt  to  account  for  the  products  directly,  the 
chemists  tried  to  so  write  the  reaction  as  to  account  for  all  the 
experimental  facts.  Now  the  attempt  to  derive  the  four  products 
in  a  quantitative  manner  from  the  molecule  of  sugar  was  enough 
to  exhaust  the  patience  even  of  a  chemist  of  the  analytical  type, 
and  numerous  different  equations  were  the  result.  Pasteur,  for 
example,  wrote  the  reaction  :  98  glucose  -f-  60  water  =  24  sue- 


VOL.  1]  Taylor — On  Fermentation.  209 

cinic  acid  -\-  144  glycerine  -(-  60  carbon  dioxide,  and  according 
to  him  this  reaction  occurred  in  4  or  5  per  cent,  of  the  substrate, 
while  the  rest  was  fermented  entirely  to  alcohol  and  carbon  diox- 
ide. That  these  bodies  might  have  nothing  to  do  with  the  alco- 
holic fermentation  per  se,  that  they  might  be  secondary  products, 
was  suggested,  but  rejected.  From  the  biological  point  of  view 
this  was  natural ;  from  the  chemical  point  of  view  it  was  illogical. 
We  know  now,  from  the  studies  on  zymase,  that  glycerine  and 
succinic  acid  are  not  products  of  alcoholic  fermentation  at  all, 
and  do  not  occur  in  a  pure  zymase  fermentation.  These  were 
apparently  metabolic  products  of  the  yeast  cells,  and  had  noth- 
ing more  to  do  with  the  fermentation  than  the  excretion  of  uro- 
bilin  has  to  do  with  the  carbon  dioxide  output  of  the  respiration. 
The  matter  is  of  importance  in  connection  with  the  question  of 
the  specificity  of  ferment  action.  The  glycerine  is  in  all  proba- 
bility derived  from  the  cleavage  of  the  fats  of  the  yeast  germs. 
That  it  does  not  come  from  the  sugar  has  been  made  very  prob- 
able by  some  ingenious  experiments  of  Rodriques  Carrando. 

Kinetics  of  the  reaction  of  alcoholic  fermentation.  The  earl- 
iest attempts  at  the  study  of  the  kinetics  of  alcoholic  fermenta- 
tion were  made  by  Dumas,  who  observed  that  the  period  of  fer- 
mentation with  a  constant  initial  inoculation  of  yeast  was  in 
general  proportional  to  the  amount  of  sugar.  Cochin  next  meas- 
ured the  reaction  by  estimating  the  carbon  dioxide.  His  figures 
were  quite  irregular.  Brown  did  some  further  tests,  and  reduced 
his  figures  to  a  curve,  which  fell  away  from  the  line  of  the  loga- 
rythmal  curve.  If  brewers  made  regular  observations  of  tem- 
perature, alcohol  content,  and  duration  of  the  fermentation,  this; 
data,  repeated  in  numerous  instances,  together  with  the  known 
•  mass  of  sugar  and  yeast  employed,  would  in  the  course  of  years 
probably  lead  to  invaluable  results,  more  valuable  surely  than, 
are  to  be  obtained  in  a  few  studies  with  zymase  fermentation. 

The  Buchners  did  not  carry  out  detailed  experiments  de- 
signed to  determine  the  quantitative  relations.  They  reached  the1- 
general  conclusion  that  the  yield  in  products  in  a  unit  of  time 
was  proportional  to  the  concentration  of  the  substrate.  At  low 
concentrations  numerous  fluctuations  were  determined.  Mac- 
fa  dy  en,  Morris,  and  Rowland  did  not  obtain  the  same  result,  but 


210  University  of  California  Publications.      [PATHOLOGY 

a  repetition  of  the  experiments  by  the  Buchners  confirmed  their 
earlier  findings.  The  same  rule  was  noted  for  the  relation  of 
velocity  to  concentration  of  ferment;  this  was  usually  found  to 
be  roughly  proportional.  At  very  high  dilutions  of  ferment  the 
results  were  often  very  conflicting.  This  was  in  all  probability 
due  to  the  varying  stability  of  the  ferment,  to  the  glycogen- 
content,  and  to  the  fact  that  the  digestion  by  the  endotryptase 
was  accelerated  in  dilute  solutions.  The  interesting  observation 
was  then  made  that  at  these  high  dilutions  the  integrity  of  the 
ferment  could  be  maintained  by  the  addition  of  egg  albumen. 
The  protecting  action  of  this  protein  the  Buchners  referred  to 
the  colloidal  qualities  of  the  egg  albumin,  and  they  brought  the 
phenomenon  in  parallel  with  the  physical  observation  of  the  sup- 
porting action  that  colloids  of  the  same  electrical  sign  display  in 
mixed  suspensions.  This  explanation  is  very  probable;  but  it  is 
also  possible  that  the  egg  albumin  acts  by  binding  the  endo- 
tryptase. Although  specific  studies  were  not  published  illustrat- 
ing the  stability  of  the  ferment  during  the  reaction  of  fermenta- 
tion, the  general  impression  derived  from  the  perusal  of  the 
Buchner  work  is  that  the  ferment  was  not  well  preserved  during 
the  course  of  a  fermentation. 

Herzog  next  studied  the  fermentation  of  glucose.  He  em- 
ployed a  powdered  yeast  that  had  been  killed  by  acetone.  Such 
an  aceton-yeast  preserves  its  activity  for  a  long  time,  but  could 
not  be  expected  to  yield  as  good  results  as  the  extract.  Never- 
theless the  results  were  quite  regular,  since  he  employed  but  one 
preparation,  and  made  sure  that  it  was  a  homogeneous  mixture. 
He  found  that  different  preparations  displayed  widely  varying 
degrees  of  glycogen  content  and  fermentative  activity,  and  that 
all  preparations  were  rapidly  inactivated  in  solution.  This  in- 
activation  he  determined  was  due  to  the  destruction  of  the  fer- 
ment by  autodigestion ;  indeed  he  went  so  far  as  to  state  that 
fermentation  may  not  continue  after  the  completion  of  auto- 
digestion.  Antiseptics  were  not  employed,  as  the  author  was 
convinced  that  but  few  bacteria  could  develop  in  the  presence  of 
the  yeast  powder,  to  which  he  ascribed  an  antiseptic  action.  He 
employed  normal  solutions  of  d-glucose  and  d-laevulose;  the  re- 
action was  measured  by  collection  of  carbon  dioxide.  The  tern- 


VOL.  1]  Taylor — On  Fermentation.  .      211 

perature  was  from  24°  to  28°  ;  no  attempt  was  made  to  maintain 
a  constancy  in  reaction.  Fairly  constant  results  were  obtained 
with  from  1  to  2  per  cent,  concentrations  of  the  ferment  in  the 
practically  normal  solution  of  sugar;  more  diluted  quantities  of 
ferment  gave  very  irregular  reactions,  especially  at  the  higher 
temperatures.  The  results  of  a  dozen  series  seemed  to  indicate 
that  the  reaction  follows  the  ordinary  logarythmal  curve;  the 
values  were  often  irregular,  and  a  tendency  to  an  increase  in 
the  value  of  the  constants  with  the  progress  of  the  reaction  was 
definitely  apparent  in  many  of  the  series.  When  calculated  ac- 
cording to  the  Henri  empiric  equation  for  an  auto-catalytic  re- 
action, the  constants  were  less  satisfactory;  not  only  were  the 
fluctuations  greater,  but  there  was  a  tendency  to  a  reduction  in 
the  value  of  the  constants,  and  these  reductions  were  more  pro- 
nounced than  were  the  increases  in  the  case  of  the  constants 
calculated  according  to  the  regular  equation.  Although  Herzog 
did  not  enter  into  the  question  of  an  interpretation  of  these  re- 
sults, it  seems  probable  that  a  progressive  inactivation  of  the  fer- 
ment (due  to  destruction  and  not  to  influence  of  products  upon 
transformation)  was  opposing  an  increase  by  auto-catalysis,  and 
the  results  represent  a  balance  between  these  factors  as  applied 
to  the  march  of  a  simple  monomolecular  reaction.  Herzog  found 
that  the  initial  rate  of  transformation  with  a  constant  ferment 
was  in  general  proportional  to  the  concentration  of  the  substrate. 
He  found  also  that  the  rate  of  fermentation  with  constant  con- 
centration of  substrate  was  proportional  to  the  square  of  the 
quantity  of  ferment,  and  that  the  increased  velocity  on  increase 
of  temperature  was  in  accordance  with  the  van't  Hoff-Arrhenius 
equation  for  such  increases  in  general  reactions.  Herzog  was 
not  satisfied  with  the  experiments,  and  he  himself  stated  only 
what  every  reader  must  feel  on  studying  the  conditions  and  re- 
sults of  the  experiments.  It  will  be  necessary  to  secure  a  greater 
purity  in  the  ferment  before  definite  results  can  be  obtained ;  the 
march  of  the  fermentation  of  the  sugar  seems  entirely  too  de- 
pendent upon  the  march  of  the  digestion  of  the  ferment  by  the 
endotryptase  and  upon  the  glycogen  in  the  yeast.  Herzog 's  re- 
sults were  indeed  much  better  than  will  be  often  obtained  by  any 
one  who  will  repeat  the  experiments,  as  I  can  testify.  With  no 


212  University  of  California  Publications.      [PATHOLOGY 

ferment  with  which  I  have  worked  have  slight  deviations  in  the 
conditions  exerted  so  bizarre  an  influence  upon  some  one  of  the 
variables  concerned.  For  myself,  I  have  become  convinced  that 
to  work  successfully  with  a  yeast  powder  (and  probably  with  an 
expressed  zymase)  one  must  have  cultivated  the  yeast  for  several 
generations  and  fixed  its  activity,  or,  to  use  a  common  expression 
with  bacteriologists,  heightened  and  standardized  its  virulence. 
Certainly  the  commercial  Sachromyces  Cerevisea  of  this  city  is 
worthless  for  the  preparation  of  a  yeast  powder:  the  glycogen- 
content  is  high,  the  proteolytic  ferment  active,  the  zymase  weak. 
This  experience  is  of  course  not  new.  Several  of  the  chemists 
and  bacteriologists  who  attempted  to  repeat  the  original  experi- 
ments of  Buchner  failed  entirely,  and  even  the  discoverers  have 
at  times  failed,  especially  with  yeast  taken  from  the  later  stages 
of  a  fermentation.  The  experience  of  brewers  has  long  been 
that  even  under  the  most  favorable  conditions  a  certain  race  of 
yeast  will  become  weakened,  and  Hayduck  has  described  a 
method  of  "regenerating"  such  a  yeast  by  cultivating  it  upon 
a  medium  rich  in  sugar  but  poor  in  nitrogen.  Green  has  sug- 
gested that  the  secretion  of  zymase  may  be  intermittent.  Thus 
far  it  has  not  been  possible  to  modify  at  will  the  zymase  produc- 
tion of  a  certain  race  of  yeast,  but  by  careful  cultivation  at  low 
temperature  something  may  be  accomplished.  The  same  expe- 
rience is  sometimes  made  with  pathogenic  microorganisms.  While 
it  is  usually  possible  to  restore  the  virulence  of  such  a  microor- 
ganism by  passing  it  through  an  appropriate  animal,  sometimes 
the  culture  has  become  so  altered  by  prolonged  cultivation  upon 
artificial  media  that  it  is  difficult  to  restore  it  to  virulence.  What 
we  do  not  know  is:  what  are  the  particular  factors  that  deter- 
mine the  degree  of  production  of  zymase  and  endotryptase  re- 
spectively? In  my  experience,  which  has  been  solely  upon  local 
material  and  entirely  unsatisfactory,  the  proteolytic  ferment  has 
been  very  disproportionately  active.  And  even  with  rather  high 
concentration  of  the  ferment  the  results  have  always  been  so  ir- 
regular as  to  resemble  those  experiments  first  done  by  the  Buch- 
ners  and  repeated  by  many  others,  in  which  with  high  dilutions 
of  an  otherwise  active  ferment  the  results  become  entirely  irreg- 
ular and  incapable  of  any  interpretation. 


VoL-  !]  Taylor. — On  Fermentation.  213 

Herzog  has  since  reported  the  results  of  a  second  study  of 
alcoholic  fermentation,  using;  zymase.  His  results  were  very  ir- 
regular ;  he  did  not  succeed  in  securing  a  zymase  sufficiently  rich 
in  the  alcoholic  ferment  and  poor  in  glycogen  and  endotryptase 
to  warrant  the  interpretation  of  the  results  from  the  kinetic  point 
of  view.  Different  preparations  of  zymase,  as  first  pointed  out 
by  the  discoverers  themselves,  vary  widely  in  enzymic  power,  and 
it  is  to  be  expected  that  with  some  preparations  quantitative  ex- 
periments are  impossible. 

Slator  measured  the  progression  of  an  alcoholic  fermentation 
by  the  estimation  of  the  pressure  of  carbon  dioxide.  He  found 
the  transformation  quite  independent  of  the  substrate  concen- 
tration, but  closely  proportional  to  the  mass  of  ferment.  In  very 
weak  solutions  of  sugar  the  transformation  was  roughly  propor- 
tional to  the  substrate  concentration.  From  this  Slator  concludes 
that  the  reaction  is  not  one  of  the  first  order.  The  temperature 
coefficient  he  determined  to  be  high,  though  deminished  with  in- 
creasing temperature.  The  values  given  are  much  too  high  for 
diffusion  velocities. 

Gromow  has  recently  published  the  results  of  a  study  of  fer- 
mentation with  zymase,  illustrating  by  the  variations  from  the 
results  of  Herzog  the  uncontrollable  difficulties  that  attend  the 
study  of  this  ferment.  He  determined  first  that  notable  quan- 
tities of  products  were  evolved  from  the  glycogen,  which  the  ex- 
tract first  splits  and  then  ferments  to  alcohol;  and  that  the  re- 
action was  identical  in  an  atmosphere  of  hydrogen  and  of  air. 
He  made  the  new  observation  that  a  false  equilibrium  could  be 
established  in  the  system;  and  that  while  the  addition  of  more 
ferment  would  reinaugurate  the  reaction,  the  addition  of  more 
sugar  would  not.  The  addition  of  the  products  to  the  system 
early  in  the  reaction  exerted  an  accelerating  reaction ;  that  is,  in 
this  fermentation  we  have  a  tendency  to  autocatalysis.  The 
transformation  was  a  little  less  than  proportional  to  the  mass 
of  substrate,  at  high  dilutions,  but  otherwise  the  mass  of  sub- 
strate seemed  to  have  little  influence  on  the  rate  of  transforma- 
tion. The  fermentation  was  a  little  less  than  directly  propor- 
tional to  the  quantity  of  ferment.  High  concentrations  of  sugar 
were  found  to  depress  somewhat  the  proteolytic  ferment.  The 


214  University  of  California  Publications.       [PATHOLOGY 

very  interesting  observation  was  made  that  quinine  exerts  a  very 
depressing  influence  upon  the  endotryptase  at  concentrations 
that  do  not  affect  the  activity  of  the  alcoholic  ferment;  thus  it 
was  in  some  measure  possible  to  reduce  the  autodigestion  during 
the  course  of  an  experiment.  Apart  from  the  independence  of 
the  rate  of  transformation  of  the  substrate  concentration,  these 
findings  agree  better  with  our  general  conceptions  of  catalysis 
than  did  the  results  of  Herzog. 

Aberson  has  published  a  noteworthy  study  of  alcoholic  fer- 
mentation. He  found  the  transformation  quite  closely  propor- 
tional to  the  mass  of  sugar,  the  constants  tended  to  increase,  and 

a  better  concordance  was  secured  when  the  empiric  equation  of 

1      A.  I  x 
Henri  (  C  =  -  I  -    -,)  was  employed.     He  found  the  accelera- 

t          A. — OC 

tion  proportional  to  the  mass  of  ferment.  He  observed  also  un- 
der certain  circumstances  an  equilibrium,  such  as  has  been  noted 
in  many  fermentations,  and  was  able  to  obtain  data  tending  to 
suggest  an  actual  reversion.  The  data  tending  to  suggest  the 
reversion  of  the  alcoholic  fermentation  do  not,  however,  carry 
conviction.  The  reaction  is  one  that  takes  place  in  two  phases. 
It  will  be  recalled  from  the  earlier  work  of  Tammann  and 
Nernst,  that  to  accomplish  the  reversion  of  the  reaction  Zn  -)- 
H,,S04  <=>  ZnS04  -(-  H2  the  hydrogen  concentration  in  the  sys- 
tem had  to  exceed  eighteen  atmospheres  pressure.  The  reversion 
of  the  reaction  of  alcoholic  fermentation  would  in  a  similar  man- 
ner require  a  pressure  of  carbon  dioxide,  and  this  pressure  would 
need  to  be  high.  It  is  possible  that  the  ferment  might  shift  the 
point  of  equilibrium,  though  this  would  hardly  occur  to  a  very 
marked  extent. 

Eulerjias  carefully  investigated  the  alcoholic  fermentation  of 
glucose.  He  found  the  constants  when  calculated  according  to 
the  regular  equation  were  regular  and  fairly  concordant  during 
the  first  half  of  the  fermentation,  but  became  progressively  more 
irregular  during  the  last  half.  The  constants  were  not  uniform 
in  different  series  of  different  concentrations;  with  increasing 
concentration  the  velocity  was  diminished.  With  fixed  relations 
of  substrate  and  ferment  in  the  system,  the  velocity  of  reaction 
tended  to  be  directly  proportional  to  the  total  concentration. 
With  strong  ferment,  the  acceleration  was  proportional  to  the 


VOL.  1]  Tiii/lor. — On  Fermentation.  215 

mass  of  the  ferment.  Throughout  the  work,  however,  one  sees 
the  disturbances  produced  by  the  endotryptase,  and  it  seems 
probable  that  until  some  way  is  devised  to  shut  out  the  auto- 
digestion  and  also  to  secure  preparations  free  of  glycogen,  re- 
liable investigations  upon  the  kinetics  of  alcoholic  fermentation 
will  not  be  obtainable.  It  was  with  high  hopes  that  the  discovery 
of  zymase  was  greeted,  for  here,  it  was  thought,  lay  the  oppor- 
tunity for  the  dynamic  study  of  the  fermentations.  Up  to  the 
present,  however,  the  experiences  have  been  less  satisfactory  than 
with  the  diffusible  and  secreted  ferments.  In  the  yeast  cell  every 
function  is  centered  in  the  one  cell,  and  its  extract  contains  all 
its  active  properties;  while  ferments  isolated  from  individual 
parts  of  higher  plants  or  secreted  from  specialized  glands  in  ani- 
mals naturally  present  a  greater  election  and  specificity  in  their 
contents  and  functions. 

Relation  of  ferment  mass  to  acceleration.  In  the  studies  of 
Gromow,  Aberson,  and  Euler  the  acceleration  was  found  to  be 
in  general  proportional  to  the  quantity  of  ferment  in  the  system. 
It  is  to  be  expected  that  the  activity  of  the  endotrypsin  would 
make  measurements  irregular. 

Reversion  of  reaction.  Apart  from  the  qualitative  work  of 
Aberson,  I  know  of  no  published  study  of  the  reversion  of  alco- 
holic fermentation,  though  this  was  in  a  sense  anticipated  by 
Lavoisier.  As  van 't  Hoff  has  pointed  out,  such  a  reversion  would 
need  to  be  accomplished  under  pressure  of  carbon  dioxide.  What 
will  make  the  reversion  difficult  is  the  destructive  action  of  the 
alcohol  upon  the  ferment.  Since  a  long  time  would  be  required 
for  the  reversion,  the  ferment  would  be  unable  to  survive  the 
influence  of  the  alcohol. 

Influence  of  temperature.  The  influence  of  increase  in  tem- 
perature cannot  be  judged,  because  of  the  action  of  the  proteo- 
lytic  ferment.  That  the  transformation  is  increased  with  tem- 
perature is  known,  but  the  digestion  of  the  ferment  by  the  endo- 
tryptase is  also  greatly  accelerated,  so  that  the  measurements  are 
very  irregular.  The  temperature  optimum  of  alcoholic  fermen- 
tation by  zymase  was  not  definitely  determined  by  the  Buchners. 
The  relations  are  in  fact  exceptionally  intricate  on  account  of 
the  presence  of  the  proteolytic  ferment.  As  low  a  temperature 


216  University  of  California  Publications.       [PATHOLOGY 

as  16-18°  seems  the  most  favorable,  though  many  facts  indicate 
that  were  the  zymase  freed  of  the  endotrypsin,  the  temperature 
optimum  would  be  much  higher.  The  findings  are,  furthermore, 
inconstant  for  different  concentrations  of  substrate  and  of  fer- 
ment. 


TOL.  l]  Taylor. — On  Fermentation.  .     217 


LITERATURE. 

Amylase. 

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Jour.  Chem.  Soc.,  35,  596. 

Brown  &  Morris.    Jour.  Chem.  Soc.,  55,  462-69,  709. 
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Henri."    Ibidem.,  pp.  118. 
Henri.3    Ibidem.,  pp.  76. 
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Musculus.    Zeitschr.  physiol.  Chem.,  2,  188. 
Pawlow.    Die  Arbeited  der  Verdauungsdrusen,  1898. 
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Eoux.    C,  r.  Acad.  Sc.,  140,  943. 
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Invertase. 

Acree  &  Hinkins.    Am.  Chem.  Jour.,  28,  370. 

Armstrong.1    Proc.  Eoy.  Soc.,  75,  500,  516. 

Armstrong.2    Ibidem.,  75,  526,  74,  195. 

Armstrong.3    Ibidem.,  75,  500,  516. 

Armstrong.4    Ibidem.,  76,  506,  592. 

Bardendrecht.     Zeit.  physik.  Chem.,  49,  456. 

Bourquelot.    C.  r.  Acad.  Sc.,  126,  1045-155,  690-155,  399-156,  762. 

Brochin.    Jour.  d.  Phar.  et  Chim.,  2C,  300. 


218  University  of  California  Publications.       [PATHOLOGY 

Brown.     Trans.  Chem.  Soc.,  81,  373. 

Brown  &  Glenclenning.    Jour.  Chem.  Soc.,  1902,  388. 

Duelaux.     Ann.  Pasteur.,  12,  96.     Traite  de  Microbilogie,  II. 

Dunstan.     Zeitschr.  f.  physik.  Chem.,  49,  59;  51,  732. 

Ecllefsen.     Muench.  Med.  Wochser.,  1904,  1585. 

Emmerling.1    Berichte,  34,  600,  2206,  3035. 

Euler.     Zeitschr.  physik.  Chem.  36,  641;  40,  498. 

Fawsitt.     Zeitschr.  physik.  Chem.,  48,  259. 

Fischer.     Zeitschr.  physiol.  Chem.,  26,  81. 

Fischer  &  Armstrong.    Berichte,  35,  3151. 

Hafner.     Zeitschr.  physiol.  Chem.,  42,  1. 

Henri.4     Zeitschr.  physiol.  Chem.,  59-194;  51,  19. 

Henri.5    Lois  generates  des  Diastastes,  1903. 

C.  r.  Soc.  BioL,  57,  171. 
Herzog.     Zeitschr.  physiol.  Chem.,  41,  416-43,  222. 

Zeitschr.  allgem.  Physiol.,  4,  163. 
Hill.     Jour.  Chem.  Soc.,  73,  638. 

Jour.  Chem.  Soc.,  83,  578. 

Jodblauer  &  Tappeiner.     Muench.  Med.  Wochsch.,  1905,  1906. 
Kastel  &  Clark.    Amm.  Chem.  Jour.,  30,  422. 
Knoblauch.     Zeitschr.  physik.  Chem.,  22,  268. 
Lobry  de  Bruyn  &  van  Erenstein.    Berichte,  28,  3078,  3085. 

Eev.  Trav.  chim.  Pays-Bas.,  14-116,  203;  16-262. 
Osborne.     Zeitschr.  physiol.  Chem.,  28,  399. 
O 'Sullivan  &  Thompson.     Trons.  Chem.  Soc.,  57,  834. 
Plazak  &  Huesek.     Zeitschr.  physik.  Chem.,  47,  733. 
Eudorf.     Zeitschr.  physik.  Chem.,  43,  275. 
Sacharoff  &  Sachs.     Muench.  Med.  Wochsch.,  1905,  No.  7. 
Straub.    Arch,  exper.  Phar.  u.  Path.,  51,  385. 
Tammann.1     Zeitschr.  physik.  Chem.,  18,  426. 
Tammann.2    Zeitschr.  physiol.  Chem.,  16. 

Visser.     Dissertation,  Amsterdam,  1904.     Zeitschr.  physik.  Chem.,  52,  257. 
Wohl.    Berichte,  &?-2103. 
Wroblowsky.    Bull.  Acad.  d.  1'Acad.  d.  Sc.  Cracovie,  1901. 


Emulsine. 

Armstrong.     Proc.  Eoy.  Soc.,  73,  520,  74,  188. 

Emmerling.    Berichte,  34,  3810. 

Fischer.    Berichte,  28,  1509. 

Henri.6    Lois  gen.  d.  Dias,  101. 

Herissey.    Eech.  sur.  1 'Emulsine  Diss.,  Paris,  1899. 

Herzog.     Konikl.  Akad.  v.  Wepenschappen,  Amsterdam,  1903. 

Pottevin.    Ann.  Pasteur,  17,  31. 

Tammann.     Zeitschr.  physik.  Chem.,  13,  25-18,  426. 

Zeitschr.  physiol.  Chem.,  16. 
Visser.     Zeit.  f .  physik.  Chem.,  52,  257. 


VOL.  1]  Taylor. — On  Fermentation.  219 

Alcohol. 

Aberson.    Rev.  Trav.  chim.  d.  Pays-Bas.,  22,  78. 

Baeyer.    Berichte,  3,  73. 

Bardendrecht.    Zeit.  physik.  Chem.,  49,  456. 

Brown.    Jour.  Chem.  Soe.,  61,  368. 

Buchner  &  Hahn.     Die  zymasegaerung,  1903. 

Buchner  &  Meisenheimer.1     Berichte,  37,  417;  38,  620. 

Buchner  &  Albertoni.     Zeitschr.  physiol.  Chem.,  44,  206. 

Zeitschr.  physiol.  Chem.,  46,  136. 
Cochin.    C.  r.  Acad.  Sc.,  96,  852. 
Duclaux.    Ann.  Past.,  10,  168. 
Dumas.     Ann.  Chim.  e.  Physik.  (5),  3,  57. 
Erlenmeyer.    Jour.  Prakt.  Chem.,  71,  382. 
Euler.     Zeitschr.  physiol.  Chem.,  44,  53-45,  435. 
Gromow  &  Grigoriew.     Zeitschr.  physiol.  Chem.,  42,  229. 
Harden.1    Jour.  Chem.  Soc.,  May,  1901. 
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Harden  &  Young.    Proc.  Chem.  Soc.,  #1-189. 
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Rev.  chem.  Pays-Bas.,  14,  116,  203,  16,  262. 
M:iignon.     C.  r.  Acad.  Sc.?  140,  1063,  1124. 
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Rodrigues  Carrondo.    Rev.  Acad.  Cien.,  Madrid,  1,  217. 
See  also  Seifest  &  Reisch.    Cenbl.  f.  bakt.  u.  Parasit.,  II,  IS,  574. 
Stoklasa.    Berichte,  36,  632-38,  664. 

Beitr.  z.  chem.  Physiol.  u.  Path.,  3,  360. 

Oest.  Chem.  Zeit..  8,  273. 
.  Ber.,  36,  622 ;  38,  664. 

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Centbl.  Physiol.,  17,  465. 

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Arch.  Hyg.,  50,  165. 

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Slator.    Proc.  Chem.  Soc.,  21,  304. 
Tammann  &  Nenst.     Zeitschr.  physik.  Chem.,  9,  1. 
Wohl  &  Oesterlein.     Berichte,  34,  1139. 


220  University  of  California  Publications.       [PATHOLOGT 


THE  PEOTEOLYTIC  FERMENTS. 

The  proteolytic  ferments  are  most  widely  distributed.  They 
exist  in  all  the  invertebrates.  First  isolated  from  plants  by 
Gorup-Besanez,  they  are  now  known  to  exist  in  all  seeds  and 
grains,  and  in  many  leaves  and  roots  of  different  plants.  The 
ferments  in  the  seeds  are  usually  active  to  a  high  degree  only 
during  the  period  of  germination.  Non-germinating  seeds,  how- 
ever, contain  peptone  (Mark).  The  sap  of  certain  trees  such  as 
the  papaya  is  exceedingly  rich  in  proteolytic  ferment;  indeed 
knowledge  of  this  ferment  may  be  said  to  be  the  most  antiquated 
of  any  ferment,  since  it  was  described  by  Griffith-Hughes  as  early 
as  1750.  The  juice  of  the  fig  tree  and  its  fruit  and  the  juice  of 
the  pineapple  also  contain  active  ferments,  and  there  can  be  no 
doubt  that  were  fruits  and  vegetables  systematically  examined, 
proteolytic  ferments  would  probably  be  found  in  all.  These  fer- 
ments are  also  to  be  found  in  many  yeasts  and  moulds ;  they  are 
present  in  all  the  yeasts  of  alcoholic  fermentation,  and  probably 
constitute  an  invariable  constituent  of  the  cryptogrammes.  They 
have  also  been  so  often  found  in  all  kinds  of  bacteria,  both  saph- 
rophytic  and  pathogenic,  that  we  are  justified  in  assuming  that 
all  bacteria  possess  them.  A  perusal  of  the  descriptions  of  these 
various  proteolytic  ferments  of  vegetable  origin  would  tend  to 
the  dictum  that  they  were  all  separate  varieties ;  but  a  careful 
consideration  of  the  methods  employed  in  the  studies  will  con- 
vince one  that  as  a  matter  of  fact  the  data  are  not  sufficient  in 
quantity  and  specific  in  quality  to  warrant  any  statements  on 
classification,  though  they  present  general  properties  that  ally 
them  to  trypsin.  Vines  considers  plant  trypsin  to  be  composed 
of  two  ferments,  one  active  with  the  coagulable  and  unhydrated 
higher  proteins,  the  other,  like  erepsin,  active  in  the  cleavage  to 
amido  acids. 

Proteolytic  ferments  have  been  found,  with  few  exceptions, 
in  all  the  marine  and  terrestrial  lower  forms  of  life.    Krukenberg 


VOL.  l]  Taylor. — On  Fermentation.  221 

failed  to  find  them  in  some  coelenterates,  Cohneim  in  some  echi- 
noderms,  and  Fermi  and  Repetto  in  parasitic  vermes.  These 
negative  results  are  difficult  to  explain,  since  we  do  not  under- 
stand how  the  protein  assimilation  is  possible  without  a  ferment. 
In  all  probability,  there  are  in  these  animals  peculiarities  in  the 
secretion,  so  that  the  ferments  escaped  detection  with  the  methods 
employed. 

In  the  higher  mammalians  proteolytic  ferments  have  been  de- 
termined in  the  gastric  secretion  (long  recognized  as  a  peculiar 
phenomenon,  but  first  described  by  Spallanzani  and  Schwann)  in 
the  pancreatic  secretion  (described  by  Corvisart,  and  named  by 
Kuehne),  in  the  succus  entericus  (discovered  by  Cohnheim),  and 
in  the  tissues  and  fluids  of  the  body.  Both  pepsin  and  trypsin 
are  described  in  the  urine.  The  tissue  ferment  is  supposed  to  be 
of  the  nature  of  trypsin,  and  the  erepsin  also  resembles  this  fer- 
ment, though,  according  to  its  discoverer,  it  is  not  able  to  digest 
COM  tillable  protein,  but  is  especially  active  in  the  cleavage  of 
peptone  to  amido  acids.  The  tissue  enzymes  are  of  the  type  of 
trypsin,  and  are  currently  termed  the  uro-tryptic,  haemo-tryptic, 
hepato-tryptic,  spleno-tryptic,  etc.,  ferments.  The  proteolytic 
ferment  described  in  milk  by  Babcock  and  Russell  has  been  defi- 
nitely confirmed  by  Vandervelle,  de  Wolle,  and  Sugg;  it  is  also 
of  the  type  of  trypsin.  Vernon  has  recently  adduced  evidence 
tending  to  show  that  erepsin  is  present  in  all  tissues.  The  tissue 
ferments  that  are  active  in  the  aseptic  autolysis  of  organs  and 
tissues  yields  the  same  products  that  are  to  be  obtained  when  the 
fermentation  is  accomplished  by  pepsin  and  trypsin.  The  study 
of  the  products  of  digestion  during  recent  years  have  taught  us 
that  the  distinctions  between  pepsin  and  trypsin  lie  in  the  differ- 
ences in  the  conditions  favorable  to  their  action  and  in  the  ra- 
pidity of  their  acceleration,  not  in  the  chemical  nature  of  their 
products,  which  have  been  shown  under  proper  conditions  of  ex- 
perimentation to  be  quite  identical.  Apart  from  the  studies  of 
Cohnheim  upon  the  con  version  of  peptone  into  amido  acid  by 
erepsin.  the  studies  upon  this  ferment  as  well  as  upon  the  tissue 
ferment  have  been  so  fragmentary  as  to  be  of  little  value  in  a 
consideration  of  ferment  action  from  the  point  of  view  of  these 
lectures,  so  that  we  will  confine  ourselves  to  pepsin  and  trypsin. 


222  University  of  California  Publications.       [PATHOLOGY 

These  ferments  are  not  secreted  as  finished  enzymes,  but  in 
the  state  of  the  zymogen.  The  peptzymogen  is  converted  into 
pepsin  by  the  action  of  the  hydrochloric  acid ;  the  trypzymogen 
is  converted  into  trypsin  by  enterokinase.  They  are,  however, 
convertible  experimentally  by  extracts  of  disintegrated  cells  of 
any  kind ;  thus  extracts  of  the  powdered  gastric  mucosa  and  pan- 
creas are  active,  though  they  have  had  no  contact  with  hydro- 
chloric acid  and  enterokinase.  Larguier  des  Bancels  has  shown 
that  both  electrolytes  and  colloids  can  activate  trypsinogen.  The 
pure  secretion  of  the  pancreatic  duct  is  entirely  inert,  as  stated 
by  Haidenhain,  and  confirmed  for  the  dog  by  Pawlow  and  for 
man  by  Glaesser.  This  conversion  of  the  trypzymogen  into  tryp- 
sin really  constitutes  a  fermentation,  as  stated  by  Pawlow  and 
quantitatively  confirmed  by  Bayliss  and  Starling.  The  reaction 
may  be  stated  thus :  trypzymogen  -)-  water  —  trypsin.  The  re- 
action is  one  of  hydrolysis;  it  occurs  slowly  in  pure  water,  and 
is  accelerated  by  many  substances,  especially  by  enterokinase. 
The  current  attempts  to  define  the  enterokinase  as  a  co-ferment 
in  the  sense  of  Bertrand,  as  a  sensibilator  in  the  sense  of  Bordet, 
or  as  the  amboceptor  in  the  sense  of  Ehrlich,  do  not  rest  on  objec- 
tive grounds. 

Proteolytic  ferments  present  wide  degrees  of  variation  in  dif- 
ferent preparations.  A  comparison  of  the  different  commercial 
preparations  on  the  market  will  illustrate  this  fact  in  a  striking 
manner.  Pepsin  and  trypsin  will  present  wide  variations  when 
prepared  from  the  same  extract  by  different  methods  of  precipi- 
tation. These  variations  are  three-fold.  Firstly,  variations  in 
enzymic  strength;  different  preparations  will  vary  as  much  as 
300  per  cent.  Secondly,  variations  in  the  resistance  to  hydroly- 
sis ;  some  are  very  easily  destroyed  during  the  course  of  a  diges- 
tion, others  are  quite  resistant.  Thirdly.,  variations  in  the  tem- 
perature optimum.  Different  preparations  may  vary  as  much 
as  30°  to  45°.  This  factor  affects  of  course  both  the  enzymic 
strength  of  the  preparation  and  its  resistance  to  hydrolysis. 
When  to  these  variations  that  occur  in  the  ferment  prepared  from 
one  extract  we  add  the  varying  strength  of  the  original  extrac- 
tions or  secretions,  there  is  small  wonder  that  the  experimental 
variations  are  so  striking.  They  indicate  also  how  impossible  it 


"VOL.  l]  Taylor.— On  Fermentation.  223 

is  to  measure  the  enzymic  strength  by  any  one  method,  above  all 
by  the  Mett  method  of  linear  digestion  in  capillary  tubes. 

The  reaction  of  protein  fermentation.  The  fermentation  of 
the  albuminous  substances  is  an  act  of  hydrolysis.  As  has  long 
been  known,  all  proteins  when  heated  in  pure  water  are  more  or 
less  rapidly  hydrolyzed.  This  alone  indicates  that  at  all  temper- 
atures the  hydrolysis  is  in  slow  progress.  The  older  authors  had 
noticed  that  when  coagulable  protein  was  long  preserved  it  was 
prone  to  lose  its  property  of  coagulability.  This  slow  auto- 
hydrolysis,  which  we  may  be  certain  always  occurs  when  an  al- 
buminous body  is  suspended  in  water,  may  be  experimentally 
demonstrated,  but  since  the  velocity  is  very  low,  a  long  time  is 
required.  I  have  preserved  pure  sterile  globulin  in  distilled 
water  for  eighteen  months,  and  at  the  end  of  that  time  deter- 
mined that  not  only  had  the  degree  of  coagulability  diminished, 
but  the  globulin  could  not  all  be  precipitated  by  saturation  with 
magnesium  sulphate  or  half  saturation  with  ammonium  sulphate, 
a  portion  was  precipitable  only  by  saturation  with  ammonium 
sulphate  at  high  temperature.  Globulin  had  obviously  been  hy- 
drolyzed to  the  state  of  proteose.  I  have  also  determined  that 
leucine  may  be  recovered  from  a  sterile  solution  of  casein  (really 
a  suspension)  in  pure  water,  and  that  arginine  may  be  recovered 
from  a  solution  of  protamine  sulphate  in  pure  water — both  after 
the  lapse  of  a  year  or  more.  An  organ  may  be  sterilized  by  being 
boiled,  which  will  also  destroy  the  tissue  ferments,  and  after  the 
lapse  of  months  amido  acids  may  be  recovered  from  the  tissue. 
Pure  neucleo-proteid  may  be  preserved  stirile  in  pure  water,  and 
after  a  long  time  purin  bodies  may  be  isolated  from  the  solution. 
These  auto-hydrolyses  of  protein  constitute  the  reaction  of  which 
fermentations  as  well  as  the  acid  cleavages  constitute  the  acceler- 
ations. The  agent  of  this  aiito-hydrolysis  we  assume  to  be  the 
dissociated  hydrogen  of  water. 

The  qualitative  reactions  involved  in  the  hydrolysis  of  an 
albuminous  body  have  been  studied  in  part  through  the  aid  of 
digestions,  in  part  by  means  of  the  cleavage  with  acids.  The  re- 
lations in  the  case  of  acids  are  simpler  and  more  easy  of  control 
than  in  the  case  of  ferments,  and  the  most  recent  evidence  is  that 
the  results  are  in  both  procedures  quite  the  same.  In  considering 


224  University  of  California  Publications.       [PATHOLOGY 

the  hydrolysis  of  protein,  it  must  be  recalled  that  we  are  lim- 
iting our  consideration  to  what  we  term  common  protein,  exclud- 
ing neucleo-proteids,  glyco-proteids,  and  such  bodies  that  contain 
definite  complex  structures  not  in  themselves  protein. 

In  general  terms  the  hydrolysis  of  a  protein  may  be  said  to 
pass  through  the  following  stages :  Original  albumin,  non-coag- 
ulable  albumin,  primary  proteose  (possibly  several),  secondary 
or  deutero-proteose  (probably  several),  peptone,  an  unnamed 
sub-pepton  (possibly  identical  in  the  fermentations  with  the 
polypeptide  of  Fischer),  poly-amido-acids,  grouping  under  this 
term  the  hexon  bases,  di-  and  mono-amido-acids.  The  amido 
acids  are  not  hydrolyzed  to  lower  simpler  bodies,  but  they  may 
by  energetic  oxidation  and  reduction  be  converted  into  carbon 
dioxide  and  ammonia,  while  bacterial  oxidations  sometimes  con- 
vert them  into  basic  amines.  The  sulphur  of  the  molecule  is  con- 
tained in  the  substance  cystin,  which  is  the  disulphide  of  a-amino- 
thio-oxyproprionic  acid.  This  general  scheme  demands  a  closer 
consideration  of  the  several  steps. 

The  first  stage,  the  conversion  of  the  albumin  into  non-coag- 
ulable  protein,  is  not  observed  for  all  proteins,  but  is  encountered 
with  many  and  is  typically  represented  by  the  liquefaction  of 
fibrin.  In  all  probability  the  process  cannot  always  be  compared 
to  the  conversion  of  raw  into  soluble  starch,  or  to  the  abolition 
of  gel  properties.  In  the  case  of  gelatine,  we  know  that  a  mod- 
erately prolonged  heating  will  so  lower  the  gel-point  as  to  make 
the  preparation  a  sol  at  all  ordinary  temperatures ;  and  yet  there 
is  no  evidence  that  the  protein  has  suffered  any  chemical  change. 
The  simple  combination  of  protein  with  acid  or  alkali  will  abolish 
the  property  of  coagulation,  though  this  may  be  recovered  by 
neutralization.  Possibly  the  abolition  of  the  property  of  coagu- 
lation may  correspond  simply  to  the  disruption  of  some  chemical 
combination  favorable  to  this  property,  or  to  the  establishment 
of  some  chemical  combination  unfavorable  to  it.  In  other  in- 
stances, however,  the  alteration  is  due  apparently  to  some  change 
in  the  colloidal  properties  of  the  protein.  As  a  physical  fact,  we 
know  of  so  many  instances  of  the  conversion  of  hydro-gels  into 
hydro-sols  without  demonstrable  chemical  transformation,  that 
we  are  not  warranted  in  assuming  that  hydrolysis  plays  any- 


VOL.  l]  Taulor. — On  Fermentation.  225 

role  in  the  transformation  of  coagulable  into  non-coagulable  pro- 
tein. I  do  not  mean  by  this  to  state  that  coagulation  is  identical 
with  gelification,  but  physically  the  analogy  between  the  two  is 
sufficiently  strong  to  enforce  the  reservation  regarding  the  rela- 
tion between  hydrolysis  and  decoagulation. 

The  classification  of  the  proteoses  rests  almost  entirely  upon 
the  results  of  fractional  precipitation  with  different  salts.  This 
is  one  of  those  subjects  concerning  which  the  more  one  reads  the 
less  one  knows.  At  the  conclusion  of  the  studies  of  Kuehne  and 
his  students,  the  subject  seemed  clear;  a  little  later  Hoffmeister 
and  his  earlier  students  threw  some  confusion  into  the  classifica- 
tion. Pick,  however,  disorganized  the  Kuehne  scheme  entirely, 
and  more  confusion  has  since  been  introduced  by  Zunz  and 
Kutscher.  When  one  attempts  to  repeat  these  studies,  one  real- 
izes why  this  confusion  exists.  It  is  the  repetition  of  the  expe- 
riences with  the  dextrines.  The  numerous  investigators  have 
been  attempting  to  define  chemical  entities  under  conditions  that 
render  the  demonstration  of  such  entities  improbable  and  the 
actual  occurrence  indeed  doubtful.  For  this  there  are  two  rea- 
sons. In  the  first  place,  it  is  not  probable,  even  granting  that 
there  may  be,  let  us  say,  five  different  proteoses,  that  these  are 
formed  seriatim  and  that  their  chemical  and  physical  qualities 
are  so  distinctive  that  they  may  be  readily  separated  and  defined. 
And  secondly,  since  colloids  vary  so  widely  in  their  behavior  de- 
pending upon  their  chemical  experiences,  it  would  be  but  natural 
to  suppose  that  the  albumoses  would  exhibit  similar  variations, 
depending  upon  the  colloids  from  which  they  sprang  and  the  ma- 
nipulations to  which  they  had  been  subjected.  Precipitation  of 
these  substances  by  salts  is  less  a  chemical  than  a  physical  precipi- 
tation, in  which  the  coefficient  of  distribution  plays  a  predomi- 
nating role.  The  fractional  precipitation  of  albumoses  could  not 
therefore  be  expected  to  yield  exact  results,  since  under  variable 
conditions  of  concentrations  the  process  is  represented  by  a  gra- 
dient line.  As  a  class  proteoses  are  non-coagulable,  though  some 
of  them  incline  to  agglutination  on  cooling  of  their  hot  solutions ; 
they  yield  all  the  color  reactions  of  albumin,  are  precipitable  by 
the  heavy  metals,  but  are  much  less  sensitive  to  saline  satura- 
tions, though  they  are  all  precipitated  by  zinc  and  ammonium 


226  University  of  California  Publications.       [PATHOLOGY 

sulphates  under  appropriate  conditions.  They  do  not  diffuse, 
and  are  strictly  amorphous  bodies.  Their  molecular  weight  is 
much  lower  than  that  of  the  original  albumin. 

The  peptones  are  bodies  of  still  lower  molecular  weight ;  they 
have  some  power  of  diffusion  through  membranes ;  they  are  still 
precipitable  with  the  heavy  metals,  especially  with  compounds 
of  mercury,  iron,  tungsten,  and  wolfram.  They  respond  to  the 
Millon  and  Biuret  tests,  but  are  less  inclined  to  the  other  color 
tests  for  protein.  While  all  the  higher  proteins  are  more  or  less 
inclined  to  denaturation  on  contact  with  alcohol,  peptone  is  not 
so  affected.  Pure  solution  of  peptone  will  conduct  the  current 
appreciably,  the  first  body  in  the  series  to  do  so.  The  chemical 
combinations  of  peptone  are  more  salt-like  and  less  colloid-like 
than  the  combinations  of  the  proteoses.  The  distinction  of  two 
classes  of  peptones,  anti-  and  hemi-peptones,  has  not  been  main- 
tained. 

The  sub-peptone  stage  has  not  been  well  studied.  It  has  been 
long  observed,  but  was  first  chemically  defined  by  Fischer.1  In 
his  studies  upon  the  products  of  the  acid  hydrolysis  of  protein, 
Fischer  regularly  recovered  certain  amido-acids,  phenylalanine 
a-pyrollidin  carboxylic  acid,  which  were  not  to  be  found  among 
the  products  of  an  ordinary  tryptic  digestion.  He  then  turned 
to  the  amorphous  residue  that  remains  after  a  tryptic  digestion 
following  the  removal  of  the  amido  acids,  and  submitted  the  gum- 
like  substance  to  an  acid  hydrolysis  (after  purification  by  precip- 
itation with  phosphoralfronic  acid),  with  the  result  that  the 
missing  amido-acids  were  recovered.  Whether  this  amorphous 
substance,  which  is  surely  not  a  chemical  individual,  represents 
a  residue  of  the  protein  molecule  that  the  ferment  cannot  readily 
split,  or  whether  it  represents  a  combination  of  the  products  of 
the  digestion,  we  do  not  know.  It  is  possibly  identical  with  the 
antipeptone  of  Siegfried. 

The  poly-amido  acids  (arginine,  lysine,  and  histidine)  are 
formed  in  an  acid  hydrolysis  of  the  proteins  when  the  reaction 
is  not  too  energetic,  otherwise  di-  and  mono-amido  acids  seem 
alone  to  be  formed.  In  the  case  of  certain  simple  proteins,  the 
protamines,  the  regular  cleavage  by  acids  leads  to  these  hexon 
bases.  The  protamines  are  in  some  respects  lower  proteins  than 


VOL.  l]  Taylor. — On  Fermentation.  227 

peptone ;  they  diffuse  more  rapidly,  form  well  defined  salts  sub- 
ject to  electrolytic  dissociation,  respond  to  no  color  test  but  the 
biuret  (which  may  apply  to  an  amido  acid),  and  form  with  or- 
ganic anions  stable  physiological  compounds;  at  the  same  time 
their  molecular  weight  is  greater  than  that  of  the  lowest  peptone. 
The  poly-amido  acids  are  not  hydrolyzible  by  proteolytic  fer- 
ments, but  appear  to  be  amenable  to  other  ferments,  and  Kossel1 
has  described  in  the  liver  a  ferment  that  will  greatly  accelerate 
the  cleavage  of  arginine  into  urea  and  ornithin. 

The  mono-amido  acids  constitute  the  greater  portion  of  the 
products  of  a  protein  hydrolysis.  Ferments  act  in  this  regard  in 
general  the  same  as  acids,  only  less  energetical^.  The  statement 
is  current  that  pepsin  cannot  accelerate  the  hydrolysis  of  pep- 
tone to  amido-acids,  but  the  contrary  has  recently  been  shown 
(Langstein).  Trypsin  always  effects  the  production  of  large 
quantities  of  amido  acids,  and  the  more  these  are  studied  the 
more  nearly  the  list  approximates  that  determined  for  the  acid 
hydrolysis.  The  recent  studies  of  Emil  Fischer2  have  already  ad- 
vanced our  knowledge  of  the  final  products  of  protein  hydrolysis. 
Through  the  discovery  and  elaboration  of  an  ingenious  method 
for  the  isolation  and  separation  of  amido  acids,  he  had  been  en- 
abled to  place  these  investigations  upon  a  quantitative  plane; 
and  since  he  has  worked  with  large  quantities  of  material,  his 
results  have  for  the  first  time  exposed  a  perspective  in  this  sub- 
ject. Fischer  has  shown  that  among  the  products  of  the  acid  hy- 
drolysis of  protein  the  following  are  regularly  encountered: 
leucin,  tyrosin,  aspartic  acid,  glycocoll,  alanin,  an  amidovaleri- 
anic  acid,  glutamic  acid,  phenyl-alanine,  cystine,  serine  and  oxy- 
a-pyrollidin-carboxylic,  pyrollidin-carboxylic  acid,  and  trypto- 
phane.  In  these  substances  Fischer  and  his  students  have  been 
able  to  recover  more  than  three-fourths  of  the  nitrogen  of  the 
protein  molecule,  and  when  one  takes  into  consideration  that  the 
methods  do  not  pretend  to  be  quantitative,  one  is  driven  to  the 
conclusion  that  the  group  represents  in  all  probability  in  an  ap- 
proximate manner  the  quantitative  yield  of  the  products  of  pro- 
tein hydrolysis.  These  observations  were  made  upon  several  dif- 
ferent proteins — casein,  egg  albumin,  globulin,  edestin,  hemo- 
globin, and  fibrin.  Now  these  same  bodies  have  all  been  found 


228  University  of  California  Publications.       [PATHOLOGY 

in  tryptic  digestions,  and  nearly  all  of  them  in  peptic  digestions. 
In  both  of  these,  however,  the  hoxon  bases  appear;  in  the  acid 
hydrolysis  they  are  less  prominent.  In  the  peptic  and  tryptic 
digestions  may  be  found  in  addition  cystin,  oxyphenylaethyla- 
mine  and  pentamethylendiamine.  The  latter  two  are  probably 
derived  from  ornithin  by  bacterial  fermentation,  and  do  not  thus 
represent  primary  products  of  digestion.  It  is  more  difficult  to 
secure  a  complete  hydrolysis  with  trypsin  than  with  acids ;  it  is 
still  more  difficult  to  secure  complete  cleavage  with  pepsin,  but 
they  can  be  secured,  and  the  differences  seem  only  those  of  de- 
grees. 

These  amido  acids  have  lost  all  the  qualities  of  proteins.  They 
are  crystalline,  not  colloidal,  are  usually  basic,  form  well  denned 
salts  and  especially  good  esters,  and  conduct  themselves  in  every 
way  as  do  the  synthetic  bodies  of  the  same  composition.  Their 
constitution  and  molecular  configuration  are  in  many  instances 
not  understood.  They  contain  a  much  higher  percentage  of  nitro- 
gen than  do  the  proteins,  and  this  regular  accumulation  of  nitro- 
gen in  the  molecule  as  the  scale  is  descended  as  a  fact  of  meaning. 

The  elective  affinities  of  these  ferments  as  between  different 
proteins  in  the  same  system  have  not  been  well  investigated. 
Gompel  and  Henri  have  shown  that  if  raw  and  coagulated  egg 
albumin  are  placed  with  trypsin  in  the  same  system,  the  former 
protein  will  be  first  digested.  The  current  notion  that  raw  egg 
albumin  contains  an  anti-trypsin  is  an  error.  Pepsin  is  not  able 
to  digest  protamines,  mucin,  chitin,  or  keratine ;  trypsin  is  not 
able  to  digest  reticulin.  Pepsin  is  not  able  to  digest  the  synthetic 
polypeptides  of  Fischer;  trypsin  digests  many.  According  to 
Gonnermann,  trypsin  is  able  to  split  acetamide,  acetanilide,  and 
formanilide.  Attempts  have  been  made  to  show  that  ferments 
are  elective  towards  their  own  species,  in  the  sense,  for  example, 
that  a  trypsin  would  digest  the  casein  from  its  own  species  more 
easily  than  from  a  different  species.  These  hypotheses  have  not 
been  confirmed. 

The  modus  operandi  of  the  hydrolysis  of  protein  is  entirely 
unknown.  Information  on  this  point  is  for  the  immediate  future 
to  be  hoped  for  largely  through  the  Fischer  studies  on  the  hy- 
drolysis of  the  synthetic  peptides.  Many  of  these  substances  are 


VOL.  1]  Taylor.— On  Fermentation.  229 

digestible  by  trypsin,  none  so  far  known  by  pepsin.  Thus  far 
the  studies  of  Fischer  and  Abderhalden  have  indicated  only  a 
few  general  suggestions  bearing  upon  the  relations  of  the  reac- 
tion. They  have  found  that  there  are  structural  relations  of 
sometimes  decisive  importance.  Thus  alanyl-glycin  (CH3.CH 
(NH2).CO.NH.CH,.COOH)  is  digestible,  while  the  isomer  glycyl- 
alanine  (NH2.CH2.CO.NH.CH(CH3).COOH)  is  indigestible. 
Those  dipeptides  are  best  digested  in  which  the  alanine  figures 
as  the  acyl.  When  oxy-acids,  such  as  tyrosin,  isoserin,  and  cystin, 
are  attached  to  the  end  of  the  chain,  digestion  is  favored.  Ra- 
cemic  bodies  they  found  to  be  digested  asymetrically.  As  a  gen- 
eral rule,  the  longer  the  chain  of  amido  acids,  the  more  readily 
was  the  compound  digested.  It  must  be  confessed  that  these  facts 
throw  little  light  on  the  fermentation  of  protein,  and  none  on  the 
reaction  of  hydrolysis  itself. 

Relation  of  substrate  mass  to  reaction  velocity.  It  is  my  con- 
viction that  all  the  work  on  the  quantitative  relations  in  the  fer- 
mentation of  protein  is  of  doubtful  value.  Through  the  maze  of 
more  or  less  conflicting  data,  one  can  detect  the  main  fact  that  in 
the  center  of  the  numerous  variables  and  factors  stands  the  law  of 
mass  action  and  the  phenomena  present  in  an  approximate  way  a 
conformity  to  the  theory.  My  doubt  of  the  validity  of  the  pub- 
lished researches  is  based  upon  the  conviction  that  in  all  the 
studies  a  proper  measurement  has  been  lacking.  The  equation 
of  the  reaction  demands  as  the  first  postulate  that  the  measure- 
ment of  the  work  done  in  a  unit  of  time  shall  mark  a  unit  of 
work;  the  differential  of  transformation  and  the  differential  of 
time  constitute  the  basis  of  calculation.  In  no  work  yet  pub- 
lished, including  of  course  my  own,  has  the  method  of  measure- 
ment been  adequate  to  mark  and  determine  the  actual  transfor- 
mation. I  have  reviewed  all  these  methods,  and  the  errors  run 
from  10  to  50  per  cent.  It  will  be  of  advantage  to  review  them 
in  detail. 

Many  investigators  have  measured  the  decoagulation  of  the 
substrate.  Thus  Vernon2  measures  the  undissolved  fibrin ;  others 
have  measured  the  undissolved  coagulated  egg  albumin,  the  un- 
gelible  gelatine,  etc.  Whether  one  weigh  the  unaltered  protein, 
determine  it  by  centrifugation,  or  by  a  determination  of  the 


230  University  of  California  Publications.       [PATHOLOGY 

nitrogen,  the  analytical  error  is  large.  But  the  chief  error  lies 
in  the  assumption  that  the  ferment  first  converts  the  hydrogel 
into  a  hydrosol  as  the  first  stage  of  work,  and  that  the  next  stage 
is  not  begun  until  this  one  is  completed.  This  error  deprives  this 
method  of  measurement  of  theoretical  validity.  With  carefully 
controlled  conditions  of  work,  each  of  these  procedures  may  seem 
to  yield  fair  results,  but  when  one  compares  series  in  which  the 
different  determinations  have  been  employed,  the  fluctuations 
will  be  seen  to  be  large  and  irregular.  That  this  method  may 
be  used  for  physiological  work  is  not  to  be  denied.  Vernon  in 
particular  has  obtained  with  it  many  valuable  facts  of  physio- 
logical bearing,  but  it  is  not  adapted  to  physico-chemical  research. 

Similar  to  the  above  is  the  method  of  estimating  the  amount 
of  substrate  that  has  retained  its  coagulability  by  heat  or  precip- 
itation by  salt  (Weber  and  Thomas).  This  aims  to  measure  the 
conversion  of  globulin  and  albumin  into  their  albumoses,  under 
the  assumption  that  this  is  completely  accomplished  before  any 
transformation  of  the  proteose  is  begun.  All  that  has  been  said 
of  the  previous  method  applies  here. 

Another  method  consists  in  the  estimation  of  the  precipitable 
protein,  using  ammonium  or  zinc  sulphate,  on  the  assumption 
that  the  albumoses  may  in  this  manner  be  separated  from  the 
true  peptones  and  amido  acids.  This  is  founded  on  the  erroneous 
assumption  that  the  albumoses  may  be  thus  separated  from  the 
peptones;  the  methods  of  saline  precipitation  are  not  reactions 
of  combination,  but  are  fractional  procedures  resting  largely  on 
the  coefficient  of  distribution.  The  method  yields  contradictions 
even  in  qualitative  work.  In  addition  it  is  theoretically  an  in- 
valid method,  because  the  albumoses  are  not  converted  into  pep- 
tone en  bloc. 

In  a  similar  manner  the  formation  of  amido  acids  has  been 
utilized  as  the  measurement.  In  addition  to  the  lack  of  theoret- 
ical validity  as  a  method  of  measurement,  this  method  has  fur- 
thermore the  disadvantage  that  no  one  knows  what  the  alleged 
precipitations  for  amido  acids,  such  as  phosphowolphramic  acid 
and  tannic  acid,  will  do  in  such  a  mixture.  Nor  does  one  know 
how  much  amido  nitrogen  is  contained  in  each  protein.  Even  the 
most  careful  work  of  the  Fischer  school  has  obtained  no  results 


VOL.  1]  Taylor. — On  Fermentation.  231 

as  yet  that  might  be  used  as  a  basis  of  calculation.  With  the 
Fischer  esterification  method  better  analytical  results  could  be 
certainly  secured,  but  the  basis  of  calculation  would  be  still 
wanting. 

The  measurement  of  the  end  products  of  a  protein  digestion 
is  at  present  entirely  impossible  for  two  reasons.  In  the  first 
place,  there  is  no  known  method  feasible  for  a  fermentation  ex- 
periment. And  secondly  the  reaction  cannot  be  completed  for 
so  long  a  time  that  the  conditions  of  ferment  maintenance  are 
impossible. 

Another  method  has  been  the  method  of  estimating  the  com- 
bined acid,  as  has  been  developed  by  Vollhard.  It  has  not  been 
demonstrated  that  the  reactions  of  acid  to  substrate  is  a  stochio- 
metric  one,  and  that  the  lower  products  do  not  display  variable 
relations.  In  fact,  it  is  quite  certain  that  this  is  the  case.  I  have 
not  been  able  to  secure  concordant  results  with  the  method.  It 
lacks  obviously  theoretical  validity,  in  that  it  also  assumes  a  block 
transformation. 

Schuetz  first  introduced  polariscopy.  Here  for  the  first  time 
the  method  was  accurate  so  far  as  the  actual  reading  was  con- 
cerned. It  depends  on  the  assumption  that  the  different  proteins 
down  to  the  stage  of  peptone  have  fixed  powers  of  rotation,  and 
that  these  are  not  alterable  or  progressive.  It  must  be  borne  in 
mind  that  solutions  of  sugar  when  freshly  prepared  exhibit  often 
marked  fluctuations  in  rotation,  until  finally  the  stable  condition 
is  established.  A  similar  condition  in  the  course  of  the  protein 
digestion  would  yield  entirely  irregular  results.  The  method 
rests  upon  the  same  error  common  to  all  the  previously  men- 
tioned ones,  in  that  it  assumes  that  the  transformation  from  one 
stage  to  the  next  occurs  in  block.  How  totally  this  would  nullify 
the  results  if,  as  we  know,  this  were  not  true,  is  easy  of  illustra- 
tion. Let  it  be  granted  that  the  original  protein,  the  primary 
albumose,  the  secondary  albumose  and  the  peptone  (to  make  no 
mention  of  other  sub-products)  had  a  fixed  factor  of  rotation 
that  was  attained  as  soon  as  the  substance  was  formed.  How 
could  one  calculate  or  measure  the  transformation  in  the  system 
when  all  four  were  present  ? 

Closely  allied  to  the  measurement  by  the  spectroscope  is  that 


232  University  of  California  Publications.       [PATHOLOGY 

by  the  spectrophotometric  measurement  of  the  biuret  reaction. 
The  theoretical  validity  and  the  practical  errors  are  identical  in 
the  two  procedures. 

The  estimation  of  the  viscosity  as  a  measurement  of  the  diges- 
tion has  been  employed  by  Spriggs.  He  states  that  the  dimin- 
ution in  the  viscosity  ran  parallel  to  the  conversion  of  coagulable 
into  uncoagulable  protein.  Under  these  circumstances  it  has  the 
same  value  as  the  estimation  of  the  coagulable  protein,  but  is  sim- 
pler of  performance. 

On  entirely  empiric  grounds,  Sjoquist  and  Henri  have  em- 
ployed the  method  of  conductivity  to  measure  the  fermentation 
of  protein.  Henri  and  his  students  and  Bayless  have  obtained 
very  good  results  with  this  method,  and  when  on  the  appearance 
of  the  paper  of  Henri  in  which  the  method  was  described  I  re- 
peated the  experiments  with  gelatine,  I  obtained  constants  that 
were  within  each  series  quite  concordant.  The  method  is  obvi- 
ously based  on  the  proposition  that  the  digestion  comprehends  the 
transformation  of  a  colloid  into  an  electrolyte ;  a  non-dissociated 
into  a  dissociated  body.  To  apply  the  method  properly,  the  con- 
ductivity of  the  fully  hydrolyzed  system  should  be  determined, 
and  then  the  equation  inverted,  and  consider  under  A  the  full 
conductivity  of  the  products  and  under  x  the  increment  in  con- 
ductivity in  the  interval  of  time  t.  Of  course  one  could  compare 
the  readings  with  each  other,  as  is  commonly  done  with  the  equa- 
tion C=—  log  (~r),  nevertheless  it  were  better,  were  it 
<2 — i\  A?. 

feasible,  to  invert  the  equation.  Despite  the  apparently  good 
results,  the  method  cannot  give  satisfaction  until  the  relations  are 
worked  out.  It  may  be  assumed  that  with  each  stage  of  hydroly- 
sis of  the  protein  molecule  the  conductivity  is  augmented.  Now 
for  the  stages  protein  -  >  albumoses  -  >  peptone,  the  increment  in 
the  known  conductivity  of  the  substances  is  slight.  At  the  point 
peptone  ->  poly-amido-acid,  however,  there  is  a  heavy  jump  in 
the  dissociation.  Now  in  the  experiment  these  are  superimposed ; 
the  biuret-bearing  substances  begin  to  disappear  at  a  time  when 
primary  albumoses  are  still  in  the  system.  Under  these  circum- 
stances the  theoretical  validity  of  the  procedure  is  very  doubtful. 
To  add  to  this  doubt  is  the  fact  that  the  conductivity  in  such  a 


VOL.  1]  Taylor.— On  Fermentation.  233 

complex  system  is  not  the  function  of  the  mass  of  dissociated 
electrolytes  solely. 

Bayliss  states  that  in  the  digestion  of  gelatine  and  casinogen 
the  chief  cause  of  the  increase  in  conductivity  is  the  splitting  off 
of  inorganic  constituents  of  the  substrate  molecules,  for  the  ca- 
sinogen in  addition  the  conversion  of  organic  phosphorus  into 
inorganic  phosphates.  According  to  this,  the  digestion  of  an  ash- 
free  substrate  would  yield  little  increase  in  conductivity.  This 
is  surely  not  true  for  protamine;  there  is  a  notable  increase  in 
the  conductivity,  but  it  is  not  progressive,  rather  step-like.  If 
the  statement  of  Bayliss  be  true,  the  method  of  measurement  of 
the  electrical  conductivity  cannot  be  a  valid  method  of  measuring 
a  fermentation  unless  it  be  shown  that  the  relation  of  the  inor- 
ganic constituents  to  the  substrate  molecules  is  a  stoechimetric 
one,  and  constant  in  different  preparations  of  the  substrate.  The 
theoretically  to  be  expected  influence  of  alteration  in  viscosity  on 
the  conductivity,  Bayliss  found  to  be  trifling. 

A  theoretically  valid  and  in  all  probability  very  delicate 
method  of  measuring  the  progress  of  a  fermentation  lies  in  the 
use  of  the  differential  tensimeter.  I  have  made  a  number  of 
tests,  using  in  part  the  simple  apparatus  of  Friedenthal,  in  part 
the  complicated  apparatus  of  Smits.  Thus  far  I  have  obtained 
no  regular  results,  but  I  am  convinced  that  the  causes  of  this  are 
methodical  and  analytical,  and  not  due  to  the  unadaptation  of 
the  method.  It  is  impossible  to  attempt  the  estimation  of  the 
molecular  concentration  by  means  of  the  freezing  or  boiling  point. 

To  avoid  the  entire  disturbance  of  the  reaction  in  many  stages 
one  should  employ  a  pure  lower  protein,  or  use  as  the  substrate 
one  of  the  synthetic  peptides  of  Fischer.  My  own  studies  on 
tryptic  digestion  have  been  carried  out  with  protamine,  and  with 
the  glycyl-glycyl-tryosin.  The  results  with  the  latter  have  been 
very  unsatisfactory  for  analytic  reasons.  Some  of  the  newer 
polypeptides  of  Fischer  apparently  offer  the  ideal  substrate, 
since  they  are  asmyetric  substances,  of  which  the  cleavage  is  a 
simple  reaction,  and  with  them  the  progression  of  the  transfor- 
mation would  be  as  simple  and  as  direct  as  in  the  inversion  of 
sugar.  I  have  begun  the  study  of  these  substances,  but  have  up 
to  the  present  obtained  no  results. 


2.34  University  of  California  Publications.       [PATHOLOGY 

For  pepsin  I  am  acquainted  with  but  one  experimental  study 
carried  out  on  correct  lines,  that  of  Sjoqvist.  He  employed  an 
emulsion  of  finely  ground  coagulated  egg  albumin  as  substrate, 
was  very  careful  with  the  temperature  and  the  degree  of  acidity, 
avoided  too  high  concentrations,  and  maintained  a  regular  me- 
chanical agitation.  The  reaction  was  measured  by  centrifuga- 
tion  of  the  particles  and  the  determination  of  the  nitrogen  in 
the  clear  fluid  by  means  of  the  Kjeldahl  method,  with  corrections 
for  the  pepsin ;  he  therefore  estimated  the  rate  of  solution  of  the 
suspended  coagulated  egg  albumin.  He  used  also  the  measure- 
ment by  conductivity.  In  the  early  portion  of  the  experiments 
the  results  were  irregular,  but  soon  the  reaction  became  progres- 
sive, and  the  transformation  then  demonstrated  itself  as  propor- 
tional to  the  substrate  within  narrow  limits.  The  great  mass  of 
other  work  upon  pepsin  and  tryptic  digestion  has  been  done  with 
the  method  of  Mette,  using  capillary  tubes  of  coagulated  albu- 
min, and  under  these  circumstances  the  relations  of  velocity  to 
substrate  concentration  cannot  be  determined,  since  the  surface 
presented  to  the  ferment  remains  constant  during  the  course  of 
the  experiment,  and  the  only  alteration  in  transformation  would 
be  due  either  to  the  inactivation  of  the  ferment  or  to  the  stag- 
nation of  products  in  the  lumen.  Huppert  and  Schuetz  studied 
the  peptic  digestion  of  ovoalbumin,  and  found  the  acceleration 
under  constant  conditions  with  high  dilutions  fairly  proportional 
to  the  mass  of  the  substrate. 

Weis  (see  Euler)  has  studied  the  peptic  digestion  of  the  pro- 
tein of  the  wheat.  The  constants  were  not  closely  concordant  in 
one  series,  while  in  different  series  of  varying  substrate  concen- 
trations the  constants  diminished  as  the  substrate  concentrations 
increased. 

Victor  Henri  and  des  Bancels  have  studied  the  digestion  of 
gelatine  and  casein  by  trypsin.  They  measured  the  reaction  by 
the  increase  in  the  conductivity.  They  found  the  velocity  of  re- 
action well  represented  by  the  curve  for  the  monomolecular  reac- 
tion. The  constants  were  in  fair  agreement  during  short  tests, 
and  were  furthermore  in  good  agreement  in  series  with  different 
substrate  concentrations.  The  products  were  found  to  depress 
the  reaction  to  a  slight  extent,  but  the  ferment  was  not  inacti- 


. 1]  Taylor. — On  Fermentation. 

vated.  The  chief  objection  to  this  work  lies  in  the  brief  time 
allotted  to  the  experiments,  usually  not  over  one  hour.  Now 
there  is  no  such  thing  as  the  digestion  of  the  casein  or  gelatine 
within  one  hour ;  the  best  that  can  be  hoped  for  within  that  time 
is  the  conversion  of  the  gellible  into  the  ungellible  form  of  gela- 
tine. The  results  of  Henri  cannot  be  obtained  with  ash-free  gela- 
tine. My  own  results  on  this  point  are  confirmed  in  the  state- 
ment of  Bayliss  previously  quoted,  that  the  increase  in  the  con- 
ductivity is  due  to  the  splitting  off  of  the  inorganic  constituents 
of  the  common  gelatine.  Under  these  circumstances,  it  is  clear 
that  the  results  of  these  investigators  may  be  related  not  to  the 
hydrolysis  of  the  protein  at  all,  but  to  the  cleavage  of  some  salt- 
gelatine  complex.  This  would  be  interesting  indeed,  but  it  would 
not  constitute  a  contribution  to  the  fermentation  of  gelatine. 

Bayliss  has  recently  published  an  elaborate  study  of  tryptic 
fermentation.  He  employed  as  substrate  gelatine  and  casinogen. 
As  method  of  measurement  he  made  use  of  the  conductivity 
method.  He  used  fresh  pancreatic  juice,  and  also  commercial 
preparations  of  trypsin.  He  found  the  constants  calculated  ac- 
cording to  the  monomolecular  equation  to  fall  rather  rapidly  as 
the  digestion  proceeded.  When,  however,  the  experiments  with 
an  excessive  substrate  concentration  are  eliminated,  the  lack  of 
concordance  in  the  constants  is  much  less  striking,  though  appar- 
ent. This  falling  off  in  the  constants  was  not  due  to  the  destruc- 
tion of  the  ferment.  Bayliss  worked  with  rather  strong  degrees 
of  alkalinity  (as  high  as  2  c.c.  -n  added  to  6  c.c.  of  system),  and 

& 

rapid  degrees  of  ferment  destruction  as  well  as  irregular  results 
might  be  expected  from  this  condition.  Tests  with  fibrin  gave 
unsatisfactory  and  uninterpretable  results.  Bayliss  studied  in 
detail  the  destruction  of  trypsin  both  in  the  digestion  system  and 
in  simple  suspension.  He  was  not  able  to  obtain  regular  results 
in  the  auto-destruction  of  trypsin,  was  convinced  that,  under  the 
conditions  of  his  tests,  it  was  slight  during  the  first  six  hours  of 
the  experiments,  and  within  that  time  did  not  retard  the  velocity. 
He  states  that  powdered  trypsin  became  weaker  with  time,  and 
quoted  the  analogous  experience  of  Tammann  with  emulsine. 
(Neither  Tammann  or  Bayliss,  however,  controlled  the  water  in 


236  University  of  California  Publications.       [PATHOLOGY 

their  preparations.  Anhydrous  preparations  of  ferments  do  not 
disintegrate  under  proper  conservation.  I  have  preparations  of 
lipase,  trypsin,  and  pepsin  three  years  old  that  are  as  active  as 
on  the  day  they  were  sealed.  But  they  must  be  free  of  water  and 
reasonable  free  of  salts.)  Bayliss  considered  the  retardation  in 
the  reaction  velocity  to  have  been  due  to  the  influence  of  the  pro- 
ducts of  digestion.  The  causes  of  this  inhibitory  influence  he 
was  not  able  to  determine  in  detail,  but  he  considered  it  due  only 
in  part  to  the  combination  of  the  products  with  the  ferment ;  he 
was  not  certain  that  some  of  the  effect  may  not  have  been  appar- 
ent only,  due  to  the  influences  of  the  products  on  the  conditions 
of  conductivity. 

Hedin  next  studied  digestion  by  trypsin.  He  employed  for 
the  substrate  casein,  serum  albumin,  and  the  white  of  egg.  He 
measured  the  reaction  by  precipitation  with  tannic  acid,  and 
the  subsequent  estimation  of  the  nitrogen  that  escaped  precipi- 
tation. In  a  word,  he  attempted  to  measure  the  sub-peptone 
nitrogen,  since  tannic  acid  is  known  to  precipitate  peptone  and 
all  proteins  high  in  the  scale.  The  chemical  validity  of  the 
method  has,  however,  not  been  established  by  Hedin,  or  by  any 
one  else.  He  assumed  that  whatever  the  actual  relations,  they 
were  constant  in  the  different  comparative  experiments.  He 
found  a  general  tendency  to  a  proportionality  betwreen  the  sub- 
strate mass  and  the  transformation,  but  the  constants  were  not 
concordant  in  different  substrate  concentrations.  As  the  sub- 
strate was  diminished,  however,  he  found  that  the  effect  per  unit 
of  casein  increased  as  the  total  amount  of  casein  diminished,  and 
finally  became  constant.  Obviously,  with  the  proper  concentra- 
tion this  means  proportionality  between  mass  of  substrate  and 
velocity  of  transformation.  He  found  that  dilution  of  the  entire 
system  had  no  effect  upon  the  total  work.  He  observed  that  the 
ferment  was  to  some  extent  inactivated  during  the  course  of  the 
digestion.  The  products  of  the  reaction  he  found  to  exert  an 
inhibitory  influence. 

Hedin  further  made  the  observation  that  the  different  cleav- 
age products  of  protein  were  not  hydrolyzed  with  the  same  readi- 
ness as  others.  If  the  ferment  concentration  be  not  excessive, 
and  under  the  assumption  that  different  constituents  of  the  pro- 


VOL.  l]  Taylor— On-  Fermentation.  237 

tern  molecule  are  digested  at  different  rates,  ''one  need  only  as- 
sume that  a  protein  molecule  of  a  certain  kind  as  well  as  a  cer- 
tain constituent  of  a  molecule  always  requires  the  same  number 
of  trypsin  time  units  for  their  digestion,  and  that  all  the  dif- 
ferent protein  molecules  present  as  well  as  the  constituents  of 
the  same  molecule  are  always  digested  in  the  same  order. ' '  That 
the  different  sub-products  might  require  different  degrees  of 
work  for  their  cleavage  is  \vell  illustrated  by  the  different  pep- 
tides  of  Fischer.  Some  of  them  are  digested  by  trypsin  with 
almost  explosive  rapidity;  others  of  analogous  chemical  compo- 
sition are  digested  with  extreme  slowness. 

In  studying  the  fermentation  of  trypsin  I  have  employed  pro- 
tamine  sulphate  from  the  salmon.  This  body,  as  pointed  out  by 
Kossel.2  is  easily  and  completely  digestible  by  trypsin,  though  not 
by  pepsin.  It  may  be  prepared  in  a  state  of  high  purity,  may 
be  sterilized  without  altering  its  characteristics,  and  may  be  ac- 
curately standardized  by  an  estimation  of  its  nitrogen.  It  has 
the  further  advantage  that  it  forms  a  comparatively  homoge- 
neous solution;  it  diffuses  through  membranes,  conducts  a  cur- 
rent measurably,  and  forms  well  defined  salts.  It  shares  with 
all  proteins  the  difficulty  of  a  direct  and  reliable  quantitative 
estimation  that  would  be  available  in  a  fermentation  experiment. 
On  hydrolysis  it  is  first  split  into  a  protone,  which  is  not  precip- 
itable  by  saline  saturation,  but  is  still  insoluble  in  acidulated  alco- 
hol, in  which  the  protamine  is  also  entirely  insoluble.  The  pro- 
ducts of  the  second  stage  of  hydrolysis  are  arginine,  with  traces 
of  a  mon-amido-valerianic  acid,  alanin  and  pyrrolidin  carboxylic 
acid ;  the  known  products  correspond  to  over  97  per  cent,  of  the 
nitrogen  of  the  protamine.  These  products  are  all  soluble  in 
acidulated  alcohol,  so  that  we  possess  a  test  that  enables  us  to 
determine  in  a  rapid  and  decisive  manner  the  end  of  the  reac- 
tion of  hydrolysis,  though  since  we  cannot  measure  the  appear- 
ance of  the  products  we  cannot  record  the  regular  march  of  the 
reaction.  In  the  study  of  the  effects  of  alkalies  and  other  sub- 
stances, as  well  as  the  relations  of  varying  concentrations  of  fer- 
ment to  the  velocity,  the  digestions  are  carried  through  to  the  end 
and  then  the  times  of  the  completed  reaction  compared.  For  the 
determination  of  the  relation  of  velocity  to  the  concentration  of 


238  University  of  California  Publications.       [PATHOLOGY 

the  substrate,  the  method  must  be  employed  differently,  since  it 
is  not  possible  to  remove  portions  of  the  material  of  a  digestion 
experiment  at  stated  times  and  determine  the  progress  of  the  re- 
action. It  is  not  difficult  to  show  that  the  reaction  is  one  of  the 
first  order.  Therefore  the  determination  of  a  constant  and  the 
relations  of  velocity  to  concentration  of  substrate  may  be  deter- 
mined by  the  preparation  of  a  series  of  a  dozen  or  more  flasks  of 
constant  volume,  reaction,  and  ferment  concentration,  containing 
of  the  substrate  respectively  10,  20,  30,  40,  50,  60,  70,  80,  90,  and 
100  milligrammes.  Now  on  the  assumption  that  the  ferment  is 
not  markedly  inactivated  during  the  short  duration  of  such  an 
experiment,  and  that  the  products  do  not  depress  the  fermenta- 
tion (which  can  be  demonstrated  for  the  concentration  em- 
ployed), it  may  be  reasonably  assumed  that  the  time  necessary 
to  digest  the  solution  with  10  milligrammes  is  the  same  as  the 
time  that  would  be  necessary  to  digest  the  last  10  milligrammes 
in  an  experiment  with  100  milligrammes,  that  the  difference  be- 
tween the  times  necessary  for  the  digestion  of  90  and  100  milli- 
grammes would  correspond  to  the  time  that  would  be  necessary 
to  digest  the  first  10  milligrammes  in  a  test  of  100  milligrammes, 

etc. ;  and  thus  one  would  be  able  to  determine  the  A  and  x  of  the 

1  A 

equation  C  =  --log  -7 — •     The  results  of  such  experiments  are 

fc  ^3. X 

given  in  the  following  tables.  The  trypsin  used  was  a  freshly 
prepared  filtered  solution  of  the  pancreatin,  according  to  Spate- 
bolz,  obtained  from  Grudbler.  This  is  not  a  particularly  active 
ferment,  but  it  is  one  of  constant  activity  for  one  bottle,  and  is 
rather  freer  of  extraneous  bodies  than  most  commercial  prepara- 
tions. On  account  of  the  daily  variations,  fresh  extracts  of  secre- 
tion cannot  be  employed  in  this  work. 

Substrate  0.150.     Vol.100.     Ferment  0.001.     T  34? 
t  15         30         45         (50         75         90        105       150 

C  (X  10-*)         68         69         69         76        66         64         62         60 

Substrate  0.100.     Other  conditions  constant. 
t  15         30        45         60         75         90        105       150 

C  (X  10-*)         79         84        80         76         73         70        64         68 

Substrate  0.075.     Other  conditions  constant. 
t  15         30         45         60         75         90        105 

C  (X  10-*)         90         97         92         88         83        88         79 


VOL.  l]  Taylor. — On  Fermentation.  239 

These  results  indicate  that  while  there  is  a  general  propor- 
tionality between  the  velocity  of  transformation  and  the  sub- 
strate concentration,  it  is  not  maintained,  but  slowly  falls.  This 
fall  cannot Jbe  corrected  by  thejose  of  the. later  Henri  equation, 
and  is  therefore  due  ta -the.  inaetivation  QJLthe  ferment. and  not 
to  a  greater  coefficient  of  combination  between  ferment  and  pro- 
duct than  of  ferment  and  substrate.  By  a  comparison  we  ob- 
serve again  the  oft  noted  fact  that  the  constants  are  not  the  same 
for  different  initial  concentrations  of  the  substrate.  In  the  essen- 
tial mathematical  sense  therefore  the  direct  proportionality  is 
only  spurious,  since  it  holds  but  for  each  particular  system.  The 
explanation  might  be  that  there  may  be  different  proportions  of 
combination  between  ferment  and  substrate,  different  valencies, 
so  to  speak,  and  that  when  the  two  are  mixed  the  reaction  pro- 
ceeds according  to  the  particular  complex  adjusted  in  that  par- 
ticular system.  It  is  to  be  noted  that  the  constants  rise  as  the 
substrate  concentration  is  reduced.  Acid  hydrolysis  of  prota- 
mine  follows  the  law  for  a  monomolecular  reaction,  that  of  sol- 
uble globuline  does  not ;  the  constants  fall  progressively. 

These  results  are  unsatisfactory  from  the  point  of  view  of  the 
theory.  Not  only  do  the  constants  not  agree  wrell  in  the  different 
measurements  in  one  series,  but  they  do  not  agree  in  different 
series  in  different  concentrations.  The  non-conformity  in  series  in 
different  concentrations  is  due  to  the  factor  of  so-called  enzvinic 
intensity.  The  lagging  of  the  constants  is  due  to  the  destruc- 
tion of  the  ferment.  The  destruction  of  the  trypsin  is  an  act  of 
hydrolysis :  trypsin  -j-  water  =  inactive  products.  This  destruc- 
tion is  more  or  less  active  is  simple  solution  in  water,  though  in 
all  probability  positive  catalysors  are  present  in  all  preparations. 
This  destruction  may  be  accelerated  by  hydrogen  ions,  hydroxyl 
ions,  and  by  colloidal  platinum.  The  proteins  protect  trypsin 
from  this  destruction,  even  in  neutral  solutions.  This  may  in 
all  probability  be  presumed  to  be  due  to  a  combination,  while  in 
the  complex  trypsin-protein,  the  hydrolysis  of  trypsin  does  not 
occur.  The  products  of  the  reaction  down  to  the  stage  of  poly- 
amido  acids  protect  also,  but  from  this  point  the  products  are 
positive  catalysors  to  the  hydrolysis  of  the  ferment.  The  hy- 
drolysis of  the  ferment  is  greatly  accelerated  by  increase  in  tern- 


240  I' niversity  of  California  Publications.       [  PATHOLOGY- 

perature.  In  view  of  these  facts,  the  mystery  which  in  the  text- 
books surrounds  the  destruction  of  ferments  should  be  expunged. 
I  have  tested  the  destruction  of  lipase,  trypsin,  and  amylase,  and 
determined  for  them  all  that  they  are  simple  hydrolyses  acceler- 
ated by  positive  catalysors  and  in  their  progress  subject  to  the 
law  of  mass  action.  Tammann  demonstrated  this  years  ago  for 
emulsine.  This  being  true,  I  have  attempted  to  apply  a  correc- 
tion for  this  destruction  of  trypsin,  using  the  equation  of  Tam- 
mann. It  will  be  recalled  that  Tammann  developed  the  equation 
under  the  assumption  that  the  transformation  of  the  substrate 
in  each  unit  of  time  would  be  proportional  to  the  substrate  and 
to  the  unaltered  ferment.  Thus -^-=0  (A — x)  (F — y) .  A  is 
the  substrate  concentration,  x  the  transformation  in  the  time  t+ 
F  the  ferment  concentration,  y  the  ferment  destroyed  in  the  time  t. 
The  reaction  for  the  hydrolysis  of  the  ferment  is  given  in  the 

equation  -^-  =  C\  (F — y) ,  which  when    integrated   under   the 

i       F 
assumption  that  y=0  when  t=0  is  £1—7  1  F_   •     When  these 

dx 
two  equations  are  combined  we  have:  -rf-  =F.C.e. — Ci.t  (A — x)  r 

e  being  2.718,  the  basis  of  the  system  of  natural  logarythms. 

When  integrated  under  the  assumption  that  x  =  0  when  /  =  #„ 

,  .A—x      c 

we  have  =1     ^-  =  ~^r.F. 

A  GI 

When  the  results  of  the  measurements  of  the  digestion  with 
protamine  with  trypsin  are  calculated  with  this  equation,  the  re- 
sults are  entirely  unsatisfactory.  The  reason  is  to  be  found  in 
two  simultaneous  tests  in  which  the  destruction  of  ferment  is 
measured  in  a  fermenting  system  and  in  simple  suspension  in 
water.  It  will  be  then  found  that  by  virtue  of  the  protective 
action  of  the  substrate,  the  destruction  is  much  more  rapid  in  the 
simple  suspension  in  water,  and  the  velocity  of  this  destruction 
cannot  be  introduced  into  the  equation. 

Further  considerations  might  at  first  suggest  that  the  ques- 
tion is  capable  of  a  further  mathematical  development.  If  it  be 
assumed  that  the  ferment  is  in  a  properly  concentrated  system 
all  combined  with  the  substrate,  it  is  obvious  that  the  ferment 
concentration  for  the  purpose  of  the  hydrolysis  of  the  ferment 
would  in  each  moment  be  inversely  proportional  to  the  sub- 
strate concentration.  As  the  line  for  A  —  x  would  be  descend- 


VOL.  l]  Taylor. — On  Fermentation.  241 

ing  in  a  logarythmal  curve,  the  line  of  ferment  concentration 
for  the  purpose  of  ferment  destruction  would  be  ascending  in  a 
logarythmal  curve.  This  curve  of  F  would  be  diminished  more 
and  more  as  the  mass  of  F  increased  and  therewith  the  velocity 
for  the  hydrolysis  of  F.  Thus  y  would  be  a  function  not  only  of 
F,  but  likewise  of  A  —  x,  since  the  mass  concentration  of  fer- 
ment for  the  purposes  of  its  own  hydrolysis  would  be  inversely 
proportional  to  A  —  x:  and  the  F^  in  each  moment  of  the  reac- 
tion would  be  a  fraction  of  F  inversely  proportional  to  the  sub- 

^ ^ 

strate  at  the  same  moment,  i.e. ,  to  A — x.    Thus  F\=F —  (f~j~ )  • 

Then  the  equation  of  Tammann  wrould  read:  ~^  =  C   (A  —  x) 

A /p  fljl' 

(F — (F  A — )  — //),  or  in  shorter  terms:  ~^  =  C  (A  —  x) 
(Fl  —  y).  By  making  a  series  of  tests  of  the  hydrolysis  of  the 
ferment  at  the  corresponding  concentrations  as  related  to  the  tn 
in  the  experimental  measurement  of  the  main  reaction,  F^  can  be 
determined  for  those  times.  When  these  corrections  are  inserted 
into  the  equation  of  Tammann,  the  results  may  be  calculated  and 
the  hypothesis  tested  by  the  uniformity  of  the  constants  for  the 
main  reaction. 

Xow  this  equation  does  not  work  in  practice.  When  the  re- 
sults are  calculated  with  it,  the  lagging  of  the  constants  is  pre- 
vented, but  for  it  is  substituted  an  acceleration  of  the  constants ; 
the  defect  is  over-corrected.  This  may  be  taken  to  indicate  that 
the  ferment  is  not  all  combined  with  the  substrate  in  such  a  sys- 
tem— a  conclusion  that  has  already  been  reached  on  different 
grounds  by  Bodenstein  and  Henri.  If  now  we  should  attempt  to 
include  the  equation  of  Henri  (described  under  intervase)  the 
proposition  that  the  ferment  combined  with  the  substrate  was 
protected  from  hydrolysis,  while  the  ferment  free  was  subject  to 
hydrolysis  and  the  ferment  combined  with  the  products  subject 
to  hydrolysis,  we  would  have  an  equation  containing  five  con- 
stants. Such  an  equation  under  the  circumstances  would  be 
valueless,  even  for  purposes  of  interpolation. 

The  remedy  lies  in  another  direction.  We  must  seek  less 
sensitive  proteolytic  ferments.  Those  of  the  higher  plants  are 
little  better  than  those  of  the  higher  animals.  In  all  probability 
in  the  lower  plants,  in  the  yeasts,  or  in  the  lower  forms  of  marine 


242  University  of  California  Publications.       [PATHOLOGY 

vegetable  or  mammalian  life,  the  proteolytic  ferments  may  be 
found  that  will  carry  on  a  simple  digestion  without  loss  of  en- 
zymic  power.  Known  for  these  ferments  already  are  their  low 
temperature  optimum  and  their  digestive  vigor.  With  the  use 
of  such  a  ferment  and  the  employment  of  one  of  the  synthetic 
asymmetrical  polypeptides  as  the  substratum,  we  may  hope  to 
show  in  an  unequivocal  manner  that  the  fermentation  of  protein 
follows  the  law  of  mass  action,  and  that  our  present  irregular  re- 
sults are  due  to  the  influence  of  uncontrollable  and  adventitious 
variables.  A  careful  comparison  of  the  results  of  Henri,  Bayliss, 
Hedin,  and  my  own  justifies  the  scepticism  expressed  in  the 
statements  introductory  to  the  discussion  of  this  subject.  I  be- 
lieve the  speculations  of  Henri,  Bayliss,  and  Hedin  will  avail 
little  until  we  secure  simpler  conditions  of  experimentation. 

Relations  of  ferment  concentration  to  rate  of  acceleration. 
This  has  been  often  studied  for  both  pepsin  and  trypsin.  The 
earliest  work,  that  of  Bruecke,  led  him  to  the  result  that  the 
velocity  of  transformation  was  proportional  to  the  quantity  of 
ferment.  Schuetz  in  his  work  found  that  the  transformation  in 
a  unit  of  time  was  not  directly  as  the  quantity  of  ferment,  but 
as  the  square  root  of  the  ferment.  For  example,  if  in  two  tests 
the  transformations  were  as  2  and  3,  the  ferment  in  the  two  tests 

were  related  as  4  to  9.  i.e.,~  =  (  „  ,'  )    .     This  relation  seems  to 

C/2  GOwo 

have  been  regularly  found  by  the  students  of  the  Pawlow  school, 
using  the  Mette  method.  Linnossier,  however,  who  used  the  same 
method,  could  not  obtain  the  same  results.  As  a  matter  of  fact, 
an  extended  experience  with  the  method  of  Mette  will  convince 
any  one  that  it  is  not  adapted  to  accurate  work,  and  that  it  is 
not  the  proper  way  of  approaching  the  problem.  Vernorr  WHS 
able  to  obtain  the  rule  in  the  digestion  of  fibrin  only  by  making- 
elaborate  and  certainly  inaccurate  corrections  for  the  inactiva- 
tion  of  the  ferment.  Sjqovist  in  the  work  on  pepsin  digestion 
found  that  the  transformation  was  proportional  to  the  quantity 
of  ferment.  The  work  of  Henri  indicates  also  that  the  transfor- 
mation is  proportional  to  the  quantity  of  ferment. 

Spriggs  studied  the  peptic  digestion  of  syntonine,  using  the 
viscometer  as  the  method  of  measurement.  His  results  were  ir- 
regular; in  some  regions  the  curves  approximated  a  proportion- 


VOL-  J]  Taylor. — On  Fermentation.  243 

ality  between  ferment  mass  and  acceleration,  in  other  regions 
they  tended  to  conform  to  the  rule  of  Schuetz.  Bayliss  and 
Hedin  determined  for  trypsin  that  the  acceleration  was  closely 
proportional  to  the  ferment  mass  if  the  ferment  concentration  be 
not  excessive.  Loehlein,  using  the  acidimetric  method  of  Voll- 
hard,  determined  the  relations  for  pepsin  to  be  proportional  to 
the  square  root  of  the  ferment  mass,  but  directly  proportional 
for  trypsin.  Huppert  and  Schuetz  were  not  able  to  obtain  good 
results  with  the  Mette  method,  but  using  the  estimation  of  the 
secondary  albumose  as  a  method  of  measurement  they  deter- 
mined that  there  was  an  approximate  proportionality  between 
the  acceleration  and  the  mass  of  ferment.  The  results  of  Pollok 
were  not  favorable  to  the  Schuetz  rules. 

In  the  digestion  of  protamine  with  trypsin  the  rate  of  trans- 
formation has  always  been  found  proportional  to  the  quantity 
of  ferment.  The  quantity  of  ferment  was  varied,  all  other  con- 
ditions being  held  constant.  The  results  are  given  in  the  follow- 
ing table,  with  the  average  error  of  the  average : 

TABLE  II. 
Digestions  at  HO-optimum.  Protamine  0.100.  Vol.  50.  £,40°. 


F. 

O.OOH 

0.006 

0.004 

0.003 

0.002 

0.001 

0.0005 

t 

37 

49 

77 

101 

145 

320 

615 

38 

50 

82 

106 

170 

340 

680 

40 

55 

82 

110 

165 

335 

654 

37 

51 

75 

102 

159 

313 

710 

35 

46 

72 

98 

147 

290 

660 

36 

47 

71 

99 

148 

280 

630 

Avg. 

37 

50 

76.5 

103 

156 

313 

657 

:±- 

0.45 

1.1 

1.9 

1.8 

2.5 

9.8 

13 

From  these  figures  it  is  apparent  that  at  this  concentration  of 
protamine  the  velocity  of  hydrolysis  is  directly  proportional  to 
the  quantity  of  ferment.  Time  X  ferment  give  the  following 
figures : 

296.  300.  309.  312.  313.  328. 

Considering  the  acknowledged  errors  in  the  experiments,  the 
concordance  is  sufficient  to  warrant  the  statement  of  direct  pro- 
portionality. The  figures  tend  to  increase  with  the  dilution  of 


-44  University  of  California  Publications.       [PATHOLOGY 

the  system,  due  to  the  destruction  of  ferment  in  the  prolonged 
periods  of  digestion. 

These  experiments  have  been  often  repeated  on  different  prep- 
arations of  protamine  with  different  preparations  of  trypsin,  and 
always  with  the  same  result.  An  interesting  method  of  illustrat- 
ing the  non-observance  of  any  such  relation  as  that  of  the  so- 
called  rule  of  Schuetz,  is  to  place  in  different  flasks  ratio  quan- 
tities of  substrate,  say  100,  200,  and  300  milligrammes,  and  after 
determination  of  the  time  required  for  the  digestion  of  the  lowest 
concentration  with  a  certain  quantity  of  trypsin,  the  others  are 
digested  with  those  quantities  of  the  ferment  as  correspond  to  the 
squares  of  the  substrates,  and  under  these  conditions  all  should 
be  digested  in  the  same  time.  The  results  always  are  that  the 
higher  concentrations  are  not  digested  in  the  same  time,  but  much 
more  quickly. 

Protamine. 

Ferment. 

t 

Protamine. 

Ferment. 

t 

As  a  physico-chemical  procedure  the  digestion  of  constant 
solutions  of  a  homogeneous  substrate,  under  constant  conditions, 
with  a  regular  measurement  of  a  unit  of  work,  employing  con- 
centrations of  substrate  known  to  lie  below  the  point  of  inacti- 
vation  of  the  ferment  by  the  products,  would  certainly  seem  to 
be  superior  to  the  digestion  of  a  tube  of  coagulated  egg  albumin. 
And  it  seems  strange  that  following  the  publications  of  Kossel 
and  Matthews,  this  procedure  has  not  been  employed  in  studies 
on  trypsin. 

I  have  studied  the  digestion  of  soluble  globuline  with  pepsin, 
and  found  the  velocity  proportional  to  the  mass  of  ferment.    The 
following  figures  will  serve  to  indicate  the  results. 
1/10%  solution  substrate 
1/100%  ferment  in  a 
1/200%  ferment  in  ft 

The  times  are  those  necessary  to  convert  all  globulin  beyond  the 
state  of  precipitation  by  magnesium  sulphate, 
a  — 86  6—182 


0.050 

0.075 

0.100 

0.002 

0.0045 

0.008 

87 

61 

53 

81 

60 

51 

0.100 

0.150 

0.200 

0.002 

0.0045 

0.008 

170 

141 

121 

156 

106 

lost. 

VOL.  l]  Taylor. — On  Fermentation.  245 

In  this  connection  it  will  be  pertinent  to  discuss  the  rule  of 
Schuetz  (according  to  which  the  accelerating  action  of  a  ferment 
is  not  proportional  to  the  mass  of  the  ferment,  but  to  the  square 
of  the  mass),  since  it  has  been  largely  in  connection  with  the  pro- 
teolytic  ferments  that  this  rule  has  been  developed.  That  this 
rule  represents  a  physico-chemical  law  of  ferment  action  per  se, 
as  is  currently  assumed  by  physiologists,  is  entirely  incorrect. 
The  fermentations  under  consideration  are  all  monomolecular 
reactions,  and  no  accelerator  of  such  a  reaction  that  acts  as  a  pure 
accelerator  and  appears  in  the  products  unchanged  and  not  in 
combination  with  or  unaltered  by  the  products  of  the  reaction 
could  have  its  influence  expressed  in  such  a  mathematical  relation 
as  is  contained  in  the  rule  of  Schuetz.  In  those  instances  of  fer- 
mentation in  which  this  rule  appears  to  be  followed,  something 
operates  to  invalidate  the  direct  proportionality  that  ought  to 
hold,  the  rule  of  Schuetz  instead  of  being  a  rule  of  ferment  action 
per  se  is  to  be  regarded  instead  as  the  result  of  some  particular 
determinant  influence  that  causes  a  regular  and  quantitative  de- 
viation from  the  normal  progression  of  the  acceleration.  For 
many  fermentations  the  rule  does  not  hold  at  all.  Thus  the  diges- 
tion of  protamine  by  trypsine,  the  fermentation  of  soluble  esters 
by  lipase,  the  action  of  rennet,  and  other  well  studied  reactions 
yield  the  normal  result  that  the  acceleration  of  the  reaction  is 
proportional  to  the  mass  of  the  ferment ;  or,  in  other  words,  the 
product  of  the  time  and  the  ferment  mass  is  constant  for  a  fixed 
concentration  of  substrate.  Most  observers  of  the  digestion  of 
the  higher  colloidal  proteins  have,  as  above  stated,  found  the  rule 
of  Schuetz  to  hold  within  certain  limits.  It  is,  however,  not  un- 
common to  find  the  rule  of  Schuetz  to  hold  for  certain  (usually 
higher)  concentrations,  while  the  rule  of  direct  proportionality 
holds  for  higher  dilutions.  Furthermore,  in  the  same  experi- 
ment, the  rule  of  Schuetz  may  hold  for  the  first  part  of  the  diges- 
tion, to  be  supplanted  later  by  a  rough  proportionality.  My  own 
studies  suggest  that  even  for  the  digestions  of  the  higher  proteins 
the  use  of  high  dilutions  will  usually  give  a  direct  proportionality 
between  degree  of  acceleration  and  ferment  mass;  the  tests  for 
the  end  reactions  are,  however,  at  these  dilutions  not  sha.rp,  and 
the  proportionality  is  only  approximate.  The  fact  that  prota- 


246  University  of  California  Publications.       |_PATHOL°GY 

mine  differs  strikingly  from  the  other  proteins  suggests  that  pos- 
sibly the  regularity  of  this  reaction  may  be  due  to  the  fact  that 
the  protamine  is  always  digested  in  the  form  of  a  salt,  usually 
the  sulphate.  Now  this  is  an  ordinary  electrolytic  salt.  After 
its  digestion,  the  products  (the  chief  one  is  arginine)  is  combined 
with  the  SO4  ion  to  form  an  electrolytic  salt.  One  might  infer 
that  the  difference  in  the  two  digestions  might  lie  in  the  fact  that 
in  the  digestion  of  the  protamine  the  products  are  completely 
combined,  whereas  in  the  digestion  of  the  higher  proteins  the 
products  remain  in  the  system  in  the  form  of  free  amido  acids, 
unbound.  That  this  cannot  be  the  direct  explanation  is  seen, 
however,  in  the  fact  that  the  situation  is  reversed  in  the  case  of 
the  digestion  of  the  different  fats.  In  the  digestion  of  the  glyce- 
rides  of  acetic  and  butyric  acids,  whose  products  are  entirely 
soluble  (and  quite  strong  acids),  the  rule  of  direct  proportion- 
ality holds ;  on  the  other  hand,  in  the  digestion  of  the  higher  fats 
whose  products  are  insoluble  (and  very  feeble  acids,  the  glyce- 
rine is  the  same  for  the  two  systems),  the  rule  of  Schuetz  holds. 
Direct  experiments  indicate  that  the  presence  of  glycerine  and 
acetic  acid  have  little  influence  on  the  action  of  lipase  from 
the  castor  bean.  One  is  therefore  led  to  wonder  if  it  is  possibly 
anything  in  the  colloid  state  that  accounts  for  the  deviation  in 
the  digestion  of  the  two  kinds  of  glycerides,  and  whether  it  may 
not  be  the  colloidal  lower  proteoses  and  not  the  amido  acids  that 
are  accountable  for  the  deviation  in  the  digestion  of  proteins. 
This  inference  could  be  tested  by  the  digestion  of  pure  peptone 
to  amido  acids,  or  by  the  digestion  of  the  polypeptides  of  Fischer. 
There  is  no  direct  data  bearing  on  occurrence  of  the  rule  of 
Schuetz,  i.e.,  on  the  occurrence  of  a  deviation  from  the  regular 
progression  of  the  digestion ;  and  the  chief  concern  it  holds  for 
us  now  lies  in  the  fact  that  it  has  been  misinterpreted  to  express 
a  rule  for  ferment  action  per  se,  and  one  that  has  been  mis- 
employed to  maintain  a  distinction  between  ferment  and  catalytic 
reactions.  Hofmeister  has  suggested  that  the  rule  expresses  a  re- 
lation of  dissociation  betwreen  the  different  components,  but  this 
has  not  been  tested  experimentally  or  mathematically. 

Relation  of  temperature  to  reaction  velocity.    The  velocity  of 
a  tryptic  digestion  is  increased  from  10°  to  40°  in  accordance 


VOL.  1]  Taylor. — On  Fermentation.  247 

with  the  theory,  being  more  than  doubled  for  every  10°.  From 
40°  to  45°  the  increase  is  much  greater  in  many  preparations, 
and  then  there  is  a  sharp  turn  in  the  curve,  as  the  destruction  of 
the  ferment  becomes  predominant.  Bayliss  found  that  trypsin 
would  act  at  zero.  In  the  investigation  of  the  influence  of  tem- 
perature, one  must  always  determine  the  temperature  optimum 
of  the  preparation,  operate  in  a  dilute  solution,  and  have  the 
system  as  nearly  neutral  as  possible,  though  still  retaining  an 
alkaline  reaction.  Henri,  Hedin,  and  Bayliss  all  found  the  same 
relation  to  increase  of  temperature,  and  I  have  always  found  it 
observed.  The  following  experiment  will  illustrate  the  facts. 

Substrates,  0.1%  protamine,  reaction  N — /10000,  trypsin  0.001. 
Temperatures  20°  30°  40° 

Times  for  complete  cleavage  1380  680  310 

The  prolonged  digestions  tend  to  lag,  a  phenomenon  due  to  the 
destruction  of  the  ferment,  even  at  the  lower  temperature. 

The  reversion  of  protein  hydrolysis  has  never  been  certainly 
accomplished.  The  phenomenon  of  plastein  formation  belongs 
here.  When  peptones  are  digested  with  gastric  contents  a  gelifi- 
cation  may  be  observed,  and  this  has  been  held  to  be  due  to  the 
reformation  of  a  globulin,  as  the  body  is  precipitated  by  carbon 
dioxide  and  by  magnesium  sulphate,  and  coagulated  by  heat  in 
the  presence  of  a  trace  of  acetic  acid.  Herzog,  who  has  recently 
repeated  the  experiments,  and  confirmed  the  experience,  with  the 
addition  of  data  showing  the  increase  in  viscosity  accompanying 
the  transformation,  is  strongly  inclined  to  regard  the  process  as 
a  reversion  of  ferment  action,  the  condensation  of  propeptone  to 
a  higher  protein. 

I  have  attempted  in  every  way  the  tryptic  and  also  the  acid 
reversion  of  the  digestion  of  protamine,  absolutely  with  no  re- 
sult. I  further  tried  to  resynthesize  6-naphtalinsulphoglycyl- 
tryosin,  the  digestible  polypeptide  recently  synthesized  by  Emil 
Fischer,  but  to  no  result.  The  reversion  of  the  fermentation 
means  simply  the  condensation  of  amido  acids  with  the  extru- 
sion of  water.  Within  the  last  few  years  this  question  has  been 
taken  up  by  Emil  Fischer,  who  has  turned  to  this  problem  the 
wonderful  synthetic  powers  that  first  accomplished  the  synthesis 
of  the  sugars,  and  then  of  the  purin  bodies.  By  the  application 


248  University  of  California  Publications.       [PATHOLOGY 

of  most  ingenious  methods,  Fischer  has  succeeded  in  condensing 
amido-acid  along  the  lines  of  the  lower  proteins.  Some  of  these 
bodies  he  terms  polypeptides,  and  they  resemble  very  strongly 
the  sub-peptone  stage  in  a  tryptic  digestion.  Many  of  these 
bodies  respond  to  the  biuret  test,  and  are  digestible  with  trypsin. 
Just  lately  it  has  been  announced  that  another  step  had  been  ac- 
complished, and  that  bodies  have  in  this  way  been  synthesized 
that  can  scarcely  be  distinguished  from  true  peptones.  Appar- 
ently we  are  on  the  verge  of  the  synthesis  of  the  higher  proteins. 
that  have  for  years  been  the  last  resort  of  the  vitalists.  It  is  only 
proper  to  state  that  Fischer  was  led  to  these  studies  by  the  re- 
sults of  his  investigations  on  the  hydrolysis  of  proteins  by  acids, 
and  the  history  of  these  studies  furnished  only  another  illustra- 
tion of  some  advantages  of  a  purely  chemical  investigation  of 
biological  problems.  It  is  the  old  story — while  the  biologist  sits 
contented,  wrapped  in  the  mantel  of  vitalism,  the  pure  scientist 
harvests  his  fields. 

While  it  is  true  that  a  reversion  of  the  hydrolyses  of  protein 
under  the  influence  of  ferments  has  not  been  demonstrable,  there 
are  indications  of  an  equilibrium  in  the  system.  No  digestion  of 
a  protein  is  completed  in  the  quantitative  sense  in  any  experi- 
ment in  vitro.  After  the  reaction  has  come  to  a  standstill,  it 
may,  under  favorable  conditions,  be  possible  to  show  that  the  fer- 
ment is  still  active.  The  further  addition  of  ferment  has  never 
in  my  experience  been  able  to  reinaugurate  the  reaction,  and  this 
is  but  a  confirmation  of  the  findings  of  Bayliss.  By  a  removal 
of  the  products,  by  the  addition  of  more  substrate,  by  raising  the 
temperature  or  by  diluting  the  system,  the  reaction  of  cleavage 
may  be  reinaugurated.  Similar  results  are  to  be  noted  in  the 
acid  hydrolysis  of  protein.  If  the  acid  be  not  too  strong,  the 
reaction  will  not  be  complete.  This  cessation  of  the  reaction  is 
not  due  to  the  binding  of  the  acid  by  the  products.  If  more  sub- 
strate be  added,  the  reaction  will  be  reinaugurated.  The  failure 
at  reversion  by  ferment  action  is  paralleled  by  failure  at  rever- 
sion by  the  aid  of  acids.  In  the  case  of  the  ferment,  the  destruc- 
tion of  the  ferment  in  the  prolonged  time  that  might  be  antici- 
pated as  necessary  for  a  demonstrable  reversion  might  be  as- 


-  1]  Taylor. — On  Fermentation.  249 

signed  as  the  cause  of  the  failure ;  for  the  failure  with  acids,  no 
such  explanation  is  at  hand. 

Conditions  of  action.  Pepsin  acts  best  in  an  acid  reaction. 
This  favorable  influence  of  acids  is  due  only  in  part  to  the  hy- 
drogen ion.  It  is  true  that  pepsin  will  act  measurably  in  any 
acid  of  appreciable  dissociation,  but  its  action  is  particularly 
stimulated  by  certain  acids,  and  without  any  relation  to  their  dis- 
sociation constants.  Hydrochloric  acid  is  most  favorable.  Next 
come  lactic  acid  and  oxalic  acid.  Sulphuric  acid  is  very  inferior 
to  hydrochloric,  phosphoric  acid  less  so.  In  fact  the  activity  of 
pepsin  is  so  connected  with  the  presence  of  acid  that  many  auth- 
ors consider  that  the  real  ferment  is  a  complex  pepsin-acid.  Of 
this  there  is  no  direct  evidence.  Other  investigators  believe  that 
the  acid  acts  upon  the  protein,  and  that  it  is  the  complex  protein- 
acid  that  is  the  substrate  of  the  digestion.  Theoretically,  the 
acid  might  be  regarded  as  a  powerful  zymo-exciter,  but  the  pecu- 
liar dependence  of  pepsin  upon  the  acid  (no  other  ferment  is  so 
dependent  upon  the  reaction  as  pepsin)  makes  this  view  seem  a 
distant  conjecture.  That  acid  alone  will  split  the  protein  is  of 
course  true,  but  the  digestion  of  protein  by  pepsin  in  an  acid  me- 
dium is  not  to  be  regarded  as  the  additive  results  of  two  cataly- 
rfors.  The  peculiarly  favorable  influence  of  hydrochloric  acid 
might,  from  the  point  of  view  of  the  acids  alone,  be  explained 
either  as  the  result  of  a  positive  influence  of  the  chloranion,  or 
as  the  result  of  negative  influences  of  the  anions  when  other  acids 
are  employed.  This  would  rest  the  positive  catalysis  upon  the 
hydrogen  ions,  and  make  the  deviations  dependent  upon  the  posi- 
tive or  negative  influences  of  the  anions  of  the  different  acid. 
The  current  statements  upon  the  most  favorable  degree  of  acid- 
ity, which  are  stated  to  be  from  one-fourth  to  three-fourths  per 
cent.,  are  of  little  value.  In  the  first  place  they  assume  that  the 
relation  is  directly  as  the  degree  of  acidity,  which  is  not  true. 
Secondly,  it  is  assumed  that  the  influence  is  related  to  the  fer- 
ment alone,  which  is  not  true.  To  determine  the  facts  two  con- 
siderations must  be  maintained.  The  substrate  concentration 
must  be  held  constant,  because  the  protein  combines  with  acids 
to  form  compounds  subject  to  a  high  degree  of  hydrolytic  disso- 
ciation. The  amount  of  acid  necessary  to  combine  with  the  sub- 


250  University  of  California  Publications.       [PATHOLOGY 

strate  must  be  determined,  and  then  the  action  of  a  constant 
quantity  of  the  ferment  may  be  determined  in  a  series  of  tests 
containing  increasing:  quantities  of  the  acid.  Hydrochloric  acid 
alone  has  been  well  studied,  and  even  here  the  data  are  not  entirely 
harmonious.  Employing  egg  albumin  and  estimating  the  diges- 
tion by  the  disappearance  of  the  power  of  coagulability,  my  own 
experience  with  human  gastric  secretion  has  yielded  the  result 
that  the  activity  of  the  fermentation  increases  up  to  about  twen- 
tieth normal,  and  then  continues  with  no  variation  up  to  consid- 
erably more  than  tenth  normal,  after  which  a  rapid  fall  occurs.. 
The  gastric  juice  of  different  individuals,  however,  often  exhib- 
ited rather  sharp  variations.  Using  commercial  pepsin  and  glo- 
bulin as  the  substrate,  I  have  found  that  rather  higher  quantities 
of  acid  may  be  tolerated,  but  this  may  have  been  due  to  the  pres- 
ence in  the  pepsin  of  protein  that  would  combine  with  acid. 
Marked  variations  were  here  also  observed  with  different  prep- 
arations. We  thus  encounter  again  the  experience  of  the  varia- 
bility of  enzymic  qualities  depending  upon  the  life  history  of  the 
preparation. 

For  trypsin  the  equally  unequivocal  statement  is  also  made 
that  it  acts  best  in  an  alkaline  medium.  In  a  restricted  sense  the 
statement  is  true.  But  the  conditions  are  so  complex  that  it  is 
doubtful  whether  the  relation  is  directly  referable  to  the  reac- 
tion. Foa  found  the  alkalinity  of  pancreatic  juice  to  be  N/10000, 
of  succus  entericus  N/70000  KOH.  That  trypsin  will  act  in  dilute 
acid  solution  is  an  old  experience ;  that  it  is  very  sensitive  to  a 
slight  excess  of  alkal  is  also  an  old  observation.  Historically,  the 
idea  that  trypsin  acts  best  in  an  alkaline  medium  was  based  in 
part  upon  the  idea  that  the  contents  of  the  small  intestines  were 
alkaline.  This  has  been  much  exaggerated.  As  a  matter  of  fact, 
the  general  reaction  of  the  ilium  is  quite  neutral.  Now  when 
one  considers  that  the  products  of  tryptic  digestion  are  largely 
alkaline,  one  is  led  to  doubt  the  degree  of  preformed  alkalinity. 
In  a  study  of  the  tryptic  digestion  of  protamine  sulphate  I  de- 
termined that  an  initial  alkalinity  of  about  one-fifteenth  hundred 
normal  constituted  the  optimum  reaction.  Since  that  time,  pur- 
suing the  matter  further  with  the  aid  of  estimations  of  the  reac- 
tion by  means  of  the  gas  cell,  I  have  learned  that  that  concen- 


VOL.  1]  Taylor. — On  Fermentation.  251 

tration  of  initial  alkali  is  most  favorable  which  is  sufficient  to 
neutralize  about  a  N/1000  acid  solution  after  neutralization  of 
the  products  of  the  hydrolysis,  which  are  slightly  acid.  This  may 
not  hold  for  other  proteins.  In  any  event  it  is  clear  that  the  opti- 
11- u in  alkalinity  is  a  very  slight  one,  and  that  here  it  is  simply 
the  hydroxyl  ions  that  act,  since  the  hydrolides  of  all  the  al- 
kaline act  in  proportion  to  their  dissociation,  except  that  barium 
depresses  strongly.  Vernon*  believes  the  protective  action  of  pro- 
tein for  trypsin  to  lie  largely  in  its  power  of  binding  alkali.  This 
is  only  in  part  true,  since  destruction  occurs  in  distilled  water. 
The  alkali  simply  accelerates  the  hydrolysis. 

The  influence  of  nearly  every  imaginable  foreign  body,  salts, 
organic  substances,  alkaloids,  have  been  tested,  these  researches 
having  been  stimulated  in  the  hope  of  determining  the  influence 
of  drugs  upon  the  digestion.  Out  of  the  mass  of  conflicting  data, 
whose  importance  has  been  highly  overestimated,  it  will  suffice 
to  state  that  most  salts  have  no  effect  in  dilute  solution,  but  are 
depressant  in  high  concentrations;  that  the  salts  of  the  heavy 
metals  tend  to  depress;  that  alkaloids,  alcohols,  and  aldehydes 
tend  to  depress;  and  that  the  purin  bodies  tend  to  accelerate. 
We  meet  here  with  the  distinction  previously  observed,  that  some 
substances  simply  inactivate  a  substance,  while  others  destroy  it. 
Thus  the  neutral  salts  simply  inactivate  pepsin,  and  its  enzymic 
power  is  restored  on  their  removal;  alkaline  salts  on  the  con- 
trary destroy  it.  The  true  value  of  this  work  is  difficult  to  esti- 
mate, but  the  human  experience  that  our  digestions  are  very 
oblivious  to  moderate  abuse  indicates  that  their  practical  appli- 
cation is  not  farreaching. 

Pepsin,  under  conditions  of  favorable  environment  in  a  diges- 
tion experiment,  is  very  resistant  to  destruction.  Trypsin  is 
much  more  sensitive.  This  means  that  the  rate  of  the  hydrolytic 
cleavage  of  trypsin  is  more  rapid  than  with  pepsin.  Under  ap- 
propriate conditions  they  will  digest  each  other.  Though  the 
proteolytic  action  of  trypsin  is  much  more  marked  and  rapid 
than  that  of  pepsin,  it  dies  out  in  .an  experiment  more  quickly. 
For  both  these  ferments,  however,  the  prolonged  experiments 
once  reported  must  have  rested  upon  a  fallacious  foundation.  It 
is  not  at  all  uncommon  to  read  in  the  older  literature  of  setting 


'2~r2  University  of  California  Publications.       [PATHOLOGY 

aside  a  digestion  experiment  for  a  year  or  more.  I  have  not 
studied  pepsin  in  this  regard,  though  I  am  certain  that  in  a  ster- 
ile mixture  it  would  not  exist  a  year.  With  regard  to  trypsin,  I 
can  state  that  direct  experiments  have  yielded  the  result  that 
with  no  commercial  preparation  of  powdered  pancreas  will  the 
ferment  activity  survive  a  few  weeks ;  usually  the  ferment  is  per- 
manently destroyed  within  a  few  days.  For  both  these  ferments 
the  rule  holds  that  they  remain  active  longer,  that  is,  resist  hy- 
drolysis longer,  when  engaged  in  digestion  or  in  contact  with 
protein  than  when  simply  suspended  in  water.  When  dessicated. 
they  may  be  preserved  almost  indefinitely,  with  no  measurable 
loss  in  activity.  They  also  bear  heating  when  dry  to  over  100°. 

The  temperature  optimum  for  both  these  ferments  runs  from 
about  40°  to  45°,  and  above  50°  there  is  a  very  rapid  destruction. 
Up  to  about  45°  the  increase  in  activity  follows  the  general  law 
for  the  increase  in  the  velocity  of  a  reaction  with  increase  in  tem- 
perature. For  different  preparations  of  the  ferments  the  rela- 
tions may  be  different,  and  especially  will  the  temperature  opti- 
mum be  changed  when  the  ferment  is  acting  under  unfavorable 
conditions.  Nothing  could  more  strikingly  illustrate  the  inde- 
pendence of  the  temperature  optimum  from  the  temperature  of 
the  animal  than  the  fact  that  the  pepsin  of  the  frog  has  the  same 
optimum  as  that  of  the  wTarm  blooded  animals.  For  fishes,  how- 
ever, Hoppe-Seyler  found  a  low  temperature  more  favorable.  As 
a  whole,  the  temperature  optimum  of  ferments  lies  considerably 
higher  than  the  body  temperature  of  the  plants  or  animals  that, 
produce  them. 

For  trypsin  higher  concentrations  of  the  ferment  yield  more 
rapid  comparative  transformations.  For  pepsin  this  is  not  true: 
dilute  solutions  give  better  comparative  digestion.  This  is  in  all 
probability  due  to  the  relations  with  the  acid.  Pepsin  is  quite- 
tolerant  of  the  presence  of  the  products  of  reaction ;  in  ordinary 
concentrations  a  peptic  digestion  proceeds  no  more  rapidly  in  a 
dyalisor  (the  acid  being  kept  constant)  than  in  a  flask.  Trypsin, 
on  the  contrary,  is  sensitive  to  the  presence  of  even  a  slight  excess 
of  the  products  of  digestion ;  a  digestion  experiment  in  a  dyalisor 
proceeds  more  rapidly  and  to  a  greater  degree  than  in  a  flask. 

Pepsin  is  quite  resistant  to  the  action  of  bacteria.     This  may 


I   UNIVERSITY 


VoL-  !J  Taylor. — OH  Fermentation.  253 

in  part  be  accounted  for  by  the  fact  that  the  acid  concentration 
in  the  case  of  peptic  digestion  is  an  unfavorable  medium  for  the 
development  of  microorganisms,  though  any  one  who  has  at- 
tempted to  check  the  bacterial  fermentation  in  chronic  gastric 
disease  by  the  use  of  hydrochloric  acid  will  soon  learn  how  weak 
is  the  anti-bacterial  action  of  this  substance.  Trypsin,  on  the 
contrary,  is  in  experiments  in  vitro  very  sensitive  to  the  action 
of  bacteria ;  to  secure  results  in  a  tryptic  digestion  the  addition 
of  an  adequate  amount  of  an  appropriate  antiseptic  is  absolutely 
essential.  Now  this  is  in  direct  contrast  to  the  natural  environ- 
ments of  the  ferments.  Pepsin  is  in  the  stomach  exposed  to  com- 
paratively few  microorganisms,  while  trypsin  in  the  intestine  is. 
surrounded  by  innumerable  bacteria  of  all  kinds,  particularly 
bacteria  of  putrefaction.  Nevertheless  the  velocity  of  tryptic 
digestion  in  the  intestine  is  such  as  to  indicate  that  the  bacteria 
exert  no  noteworthy  inhibition. 

Both  pepsin  and  trypsin  have  been  isolated  in  a  crude  state ;. 
the  best  preparations  have  been  of  pepsin  by  Pekelharing.  The 
general  methods  are  the  same.  Watery  extracts  are  prepared,  or 
the  secretions  obtained.  These  are  then  filtered,  and  the  ferment 
precipitated  by  alcohol,  and  the  colloid  washed  with  alcohol.  The 
residues  are  then  dissolved  in  water,  and  freed  of  salts  and  other 
bodies  by  dyalysis :  in  this  act  the  pepsin  suffers  but  little,  but 
unless  it  is  done  at  low  temperature  trypsin  will  be  greatly  de- 
stroyed, so  that  this  process  is  often  omitted  in  the  case  of  tryp- 
sin. The  ferment  may  be  precipitated  a  second  time  by  alcohol 
or  by  ammonium  sulphate.  The  final  preparations  are  never- 
pure.  The  purest  pepsin  of  Pekelharing  was  free  of  phosphorus, 
but  contained  both  chlorine  and  iron.  These  ferments  have  a 
percentage  constitution  similar  to  ordinary  protein.  They  dis- 
play the  reaction  of  protein,  are  coagulable  by  heat,  polarize 
light,  and  on  digestion  or  hydrolysis  yield  the  products  usually 
obtained  from  neucleo-proteids  :amido  acids,  pentose,  and  purin 
bases.  They  are  absolutely  non-diffusible,  and  although  their 
solutions  are  clear  and  colorless,  they  are  always  colloidal  and 
have  a  high  viscosity  for  their  weight  concentration.  They  are 
retained  to  a  notable  extent  by  infusorial  filters,  to  some  extent 
by  paper  filters.  The  dried  preparations  and  those  precipitated 


254  University  of  California  Publications.      [PATHOLOGY 

by  ammonium  sulphate  are  very  stable;  those  precipitated  tend 
to  display  denaturation.  Solutions  of  such  pure  pepsin  will  keep 
for  a  few  days  at  low  temperature ;  solutions  of  trypsin  decom- 
pose much  more  rapidly. 

Schrumpf  has  described  a  preparation  of  pepsin  that  gave  no 
biuret,  Millon,  picric  acid,  uranium  acetate  or  ammonium  sul- 
phate reactiors,  did  not  curdle  milk,  and  was  an  active  proteo- 
lytic  enzyme. 


VOL.  l]  Taylor. — On  Fermentation.  255 


LITERATURE. 

Babcock  and  Russell.     Cenbl.  f.  Bact.  (2),  6,  22,  45,  79. 

L.  des  Bancels.    C.  r.  Acad.  Sc.,  141,  744. 

Bayliss.    Arch.  d.  Sc.  biol.,  11,  Supp.  261. 

Bayliss  and  Starling.     Jour.  Physiol.,  30,  Ql-SS,  129. 

Cohnheim.     Zeitschr.  f.  physiol.  Chem.,  33,  9. 

Euler.     Zeitschr.  f.  physiol.  Chem.,  45,  422. 

Fermi  and  Eepetto.    Cenbl.  f.  Bact.,  31,  403  . 

Fischer.1     Zeitschr.  f.  physiol.  Chem.,  33,  157. 

Fischer."     Xuirerous  studies  in  the  Berichte  of  the  German  Chem.  Soc.  and 

in  the  Zeitschr.  f.  physiol.  Chem.  during  the  last  five  years. 
Fischer  and  Aberhalden.     Zeitschr.  f.  physiol.  Chem.,  46,  52. 
Foa.     C.  r.  Soc.  Biol.,  58,  867,  1000. 
Friedenthal.     Cenbl.  f.  Physiol.,  17,  437. 
Gies.     Am.  Jour.  Physiol.,  8,  XXXIV. 
Glaessner.     Zeitschr.  f.  physiol.  Chem.,  40,  465. 
Gompet  and  Henri.     C.  r.  Soc.  Biol.,  58,  459. 
Goiniermami.     Appotek.  Zeitung,  1902,  132. 

Arch.  f.  d.  ges.  Phys.,  89,  493;  95,  278. 
Hedin.     Jour.  Physiol.,  32,  468. 
Henri  and  des  Bancels.     C.  r.  Acad.  Sc.,  136,  1581. 

C.  r.  Soc.  Biol.,  55,  563,  787,  788,  789. 
Hueppert  and  Schuetz.    Arch.  f.  d.  ges.  Physiol.,  80,  470. 
Kossel.1    Zeitschr.  f.  physiol.  Chem.,  41,  32;  42,  181. 
Kossel  and  Matthews.     Zeitschr.  f.  physiol.  Chem.,  25,  190. 
Krukenberg.    Mittheil.  a.  d.  physiol.  Inst.  Heidelberg,  2,  1,  261,  338,  366,  402. 
Kutscher.     Zeitschr.  f.  physiol.  Chem.,  23,  115.     See  also  Folin,  ibidem,  25, 

152. 

Langstein.     Beitr.  z.  chem.  Physiol.  u.  Path.,  1,  507-#,  229. 
Linioissier.     Jour.  Physiol.  et  Path.  Gen.,  1,  281. 

C.  r.  Soc.  Biol.,  52,  288. 

Loehlein.     Beitr.  z.  chem.  Physiol.  u.  Path.,  7,  120. 
Marx.     Zeitschr.  f.  physiol.  Chem.,  42,  259. 
Nierenstein  and  Schiff.    Arch.  f.  Verdauukrank,  8,  559. 
Pekelharing.     Zeitschr.  f.  physiol.  Chem.,  35,  8. 
Pelabon.    C.  r.  Acad.  Sc.,  124,  360.     These,  Bordeaux,  1898. 
Pick.     Zeitschr.  f.  physiol.  Chem.,  24,  2.Q1-28,  219. 

Beitr.  z.  chem.  Physiol.  u.  Path.,  2,  481. 
See  also  Hart.     Zeitschr.  f.  physiol.  Chem.,  33,  347. 
Pollok.     Beitr.  z.  chem.  Physiol.  u.  Path.,  6,  95. 
Schrumpf.     Beitr.  z.  chem.  Physiol.  u.  Path.,  6,  390. 
.Schuetz.     Zeitschr.  f.  physiol.  Chem.,  9,  577. 
Siegfried.     Zeitschr.  f.  physiol.  Chen..,  35,  164. 
Sjoqvist.     Scand.  Arch.  f.  Physiol.,  5,  354. 


256  University  of  California  Publications.       [PATHOLOGY 

Smits.     Zeitschr.  f.  physik.  Chem.,  39,  289. 

Spriggs.     Zeitschr.  f.  physiol.  Chem.,  35,  463. 

Thomas  and  Weber.    Cenbl.  f.  Stoffwech.  u.  Verdauukrank,  2,  14. 

Vandervelde,  de  Waele,  and  Sugg.     Beitr.  z.  chem.  Physiol.  u.  Bath.,  5,  571. 

van't  Hoff.     Vorlesuung.  u,  theoret.  u.  physik.  Chem.,  1,  202. 

Vernon.1    Jour.  Physiol.,  32,  33. 

Vernon.2    Jour.  Physiol.,  26,  405,  28,  375. 

Vernon.3    Jour.  Physiol.,  26,  420. 

Vernon.4    Jour.  Physiol.,  2G,  412,  31,  346. 

Vines.    Ann.  Bot.,  19,  171. 

Volhard.     Zeitschr.  f .  klin.  Med.,  44,  480. 

Zunz.     Zeitschr.  f.  physiol.  Chem.,  27,  219-28,  132. 


VOL.  i]  Taylor. — On  Fermentation.  257 


THE  FERMENTATION  OF  FAT.     LIPASE. 

The  fat-splitting  enzyme,  lipase,  has  a  less  wide  distribution 
in  the  vegetable  world  than  the  ferments  of  the  carbohydrates. 
Described  first  by  Muentz,  lipase  is  now  known  to  be  present  in 
most  seeds,  resting  as  well  as  germinating.  It  does  not  seem  to 
exist  in  the  growing  parts  of  higher  plants.  It  is  found  in  most 
yeasts,  notably  in  penicillium  glaucim  and  aspergillus,  and  in 
some  bacteria,  as  the  proteus  vulgaris.  Of  pathologic  bacteria 
the  vibrio  of  cholera,  the  bacillus  typhosus,  the  colon  bacillus, 
and  the  bacillus  pyocyaneous  have  the  power  of  fermenting  fats. 
In  none  of  these,  however,  does  the  faculty  seem  prominent. 
Even  in  putrefactions,  the  organic  and  volatile  acids  are  in  large 
part  derived  from  the  protein  and  carbohydrates  rather  than 
from  the  fats ;  and  it  is  not  certain  to  what  extent  the  cleavage 
may  not  have  been  accelerated  by  alkali.  In  the  seeds  rich  in 
oils,  as  in  castor  beans,  we  find  the  highest  content  of  lipase. 

The  presence  of  lipase  in  the  pancreatic  juice  was  described 
by  Claude  Bernard,  although  he  interpreted  the  phenomenon  in- 
correctly, and  it  was  Bruecke  who  first  explained  the  reaction  in 
the  process  of  emulsification  by  the  pancreatic  juice.  Lipase  is 
now  known  to  exist  in  the  secretion  of  the  stomach  (Volhard1),. 
although  not  active  in  an  acid  medium;  in  the  succus  entericus: 
(Balribebb),  in  the  liver,  blood  serum,  and  therefore  in  all  tissues 
( Hanriot1 ) .  The  statement  is  quite  current  that  the  lipase  of  the 
liver  is  different  from  that  of  the  pancreas,  because  of  a  different 
attitude  towards  acid  and  alkali.  Lipase  is  also  known  to  exist 
in  many  of  the  lower  forms  of  life,  both  marine  and  terrestrial. 

Lipase  may  be  extracted  from  tissues  or  seeds  by  water  after 
maceration  with  quartz  sand.  It  confers  an  opalescence  upon 
the  suspension,  will  not  dyalize,  filters  poorly,  and  is  indeed  re- 
tained in  large  part  upon  the  filter,  passes  through  an  infusorial 
filter  with  great  difficulty,  and  may  be  precipitated  by  alcohol. 
It  is  quite  a  stable  ferment,  even  in  the  watery  extracts  of  mam- 
malian tissues  the  ferment  is  conserved  to  a  noteworthy  degree. 


258  University  of  California  Publications.       [PATHOLOGY 

When  dried  it  may  be  kept  indefinitely.  I  have  preparations  of 
lipase  over  two  years  old  that  are  as  active  as  the  day  they  were 
prepared.  It  is  digested  by  both  pepsin  and  trypsin,  but  is  espe- 
cially sensitive  to  trypsin.  It  is  for  this  reason  very  difficult  to 
isolate  it  from  the  pancreatic  secretion.  When  a  pancreatic  pow- 
der or  extract  is  so  prepared  as  to  conserve  its  proteolytic  fer- 
ment to  the  greatest  extent,  the  lipase  is  usually  sacrificed  en- 
tirely in  the  autodigestion.  In  only  one  of  the  commercial  prep- 
arations of  the  pancreas  have  I  been  able  to  find  any  lypolytic 
action,  and  in  this  preparation,  named  Pankreon,  this  activity 
was  but  slight.  Powders  prepared  from  perfectly  fresh  pancreas, 
on  the  other  hand,  will  display  a  high  degree  of  lipolytic  activity, 
with  a  deficiency  in  proteolytic  activity.  Most  of  the  enzyme 
seems  here  to  be  in  the  state  of  the  zymogen,  but  it  is  readily 
activated  by  the  addition  of  a  little  acid.  These  pancreas  pow- 
ders are,  however,  inactivated  in  solution  within  a  day,  in  much 
less  time  in  fact  than  the  lipases  prepared  from  the  liver  and 
blood  serum,  which  are  quite  stable. 

Lipase  is  much  more  readily  prepared  from  castor  beans  than 
from  any  other  source.  The  ferment  is  best  prepared  as  follows : 
The  beans  should  be  several  months  old,  but  not  several  years 
old,  as  is  quite  the  custom  in  the  markets.  The  shells  should  be 
removed  by  hand  without  the  use  of  water.  The  seed  is  then 
macerated  and  extracted  with  alcohol-aether  for  a  day,  and  then 
extracted  for  another  day  with  aether.  Following  the  removal 
of  the  larger  portion  of  the  fat,  the  material  is  then  ground  and 
mortared  to  the  finest  consistency  and  passed  through  a  fine 
sieve.  It  is  then  extracted  with  aether  through  several  days.  It 
is  possible  within  a  week  to  secure  the  powder  free  of  fat,  and 
this  is  desirable  in  quantitative  work.  From  the  point  of  view 
of  the  ferment,  it  were  in  all  probability  permissible  to  leave  a 
certain  amount  of  fat  in  the  power,  which  could  then  be  deter- 
mined and  the  figure  added  to  the  substrate.  Since  one  should 
employ  a  pure  fat  for  the  substrate,  it  is  advisable  to  extract  all 
the  fat.  The  material  then  presents  the  form  of  a  light  impalp- 
able powder,  not  very  hygroscopic,  and  perfectly  conservable.  It 
cannot  be  readily  weighed  on  account  of  the  magnetic  property 
that  causes  the  particles  to  fiy  apart  when  touched.  It  forms 


VOL.  l]  Taylor. — On  Fermentation.  259 

with  water  a  thick  and  quite  permanent  emulsion.     A  watery 
extract  of  this  powder  will  exhibit  lipolytic  activity,  though  to 
a  much  less  degree  than  the  powder.    The  powder  on  suspension 
in  water  will  maintain  its  lipolytic  activity  for  days  if  the  tem- 
perature be  kept  low.    I  have  made  numerous  attempts  to  isolate 
the  ferment  from  the  powder  by  precipitation  of  the  watery  ex- 
tract with  alcohol,  but  always  with  the  result  that  not  only  was 
the  ferment  weaker,  but  it  was  also  very  vulnerable  and  almost 
wholly  devoid  of  that  resistance  to  outside  influences  that  is  a 
pleasant  feature  of  the  powrdered  seed.     In  the  powder  are  of 
course  nearly  all  the  constituents  of  the  seed  except  the  fat,  and 
a  few  salts  and  lipoidal  substances  that  were  soluble  in  the  al- 
cohol and  aether.    In  this  powrder,  even  when  suspended  in  water, 
the  lipase  seems  to  be  protected,  if  the  temperature  be  kept  below 
30°.     This  lipase  works  well  at  20°,  and  as  the  proteolytic  fer- 
ment in  the  bean  does  not  seem  to  attack  it  (or  to  get  around  to 
it  after  digesting  the  numerous  other  proteins  present)  at  this 
temperature,  we  are  enabled  to  work  with  the  ferment  with  satis- 
factory results.     The  powder  contains  a  great  deal  of  protein, 
and  this  seems  to  protect  the  lipase.    Once  prepared,  the  powder 
may  be  conserved  for  years.    The  commercial  residues,  obtained 
after  compression,  are  of  less  value  and  may  be  of  no  value, 
owing  to  the  heat  developed  during  the  process  of  expression  of 
the  castor  oil.    Very  old  seeds  will  also  yield  poor  powrder;  low 
in  lipase  and  tending  to  decompose.     The  powder  should  be 
sterile.    Following  the  prolonged  extraction  with  aether  and  al- 
cohol, the  powder  is  sterile,  and  any  contamination  that  may 
lodge  during  the  process  of  sieving  and  drying  may  be  killed  by 
heating  the  dry  powder  to  100°  for  a  short  time.     Since  it  is 
possible  to  sterilize  the  fats  to  be  employed  as  the  substrate,  one 
is  freed  of  the  necessity  of  employing  antiseptics.     The  ferment 
bears  such  antiseptics  as  chloroform  well,  but  the  presence  of 
much  chloroform  disturbs  the  emulsification.     Toluol  is  the  best 
antiseptic  wThen  one  is  required.    The  castor  powder  is  not  espe- 
cially strong  in  lipolytic  activity.     I  have  seen  pancreatic  ex- 
tracts twenty  times  as  strong.    But  it  is. even  in  its  action,  con- 
stant in  results,  little  disturbed  by  extraneous  conditions  in  gen- 
eral, and  very  resistant  to  destruction.     The  use  of  this  powder 


260  University  of  California  Publications.       [PATHOLOGY 

for  the  technical  cleavage  of  fats  is  now  patented  in  Europe, 
and  in  one  of  the  trade  descriptions  of  the  process  the  statement 
is  made  that  the  powder  may  be  used  over  and  over  again.  That 
is  of  course  putting  the  case  entirely  too  strongly,  but  it  is  possible 
to  secure  a  powder  so  stable  that  one  can  carry  through  tests 
that  occupy  several  weeks  at  20°  without  any  loss  in  the  enzymic 
power — a  circumstance  of  great  importance  in  the  employment 
of  a  ferment  for  research  purposes.  For  these  reasons  I  have 
latterly  confined  myself  to  the  use  of  this  ferment,  and  have  been 
able  to  obtain  results  that  I  at  least  could  not  obtain  with  animal- 
ferments. 

This  vegetable  lipase  acts  best  in  an  acid  reaction.  The  pow- 
der itself  is  acid.  I  have  never  been  able  to  prepare  a  neutral 
powder.  The  powder  in  the  tests  to  be  described  was  acid  in  the 
proportion  1  G.  =  0.4  c.c.  N/10.  The  degree  of  the  reaction 
should  be  slight,  though  the  ferment  will  tolerate  without  inacti- 
vation  other  than  reversional  quite  a  degree  of  acidity  due  to  such 
slightly  dissociated  acids  as  acetic  and  butyric  acids ;  for  example, 
a  thirtieth  normal.  Indeed  the  products  exert  an  autocatalytic 
influence,  though  it  cannot  be  affirmed  that  this  is  due  to  the  acid 
alone.  Some  of  the  acid  is  undoubtedly  combined  with  the  pro- 
tein of  the  powder,  which  probably  explains  a  fact  that  is  to  be 
-mentioned  later,  that  this  ferment  fails  by  a  little  the  ordinary 
point  of  equilibrium  for  the  system  under  experimentation. 
Many  authors  in  writing  upon  the  favorable  or  unfavorable  in- 
fluence of  alkali  do  not  seem  to  realize  that  unless  the  alkali  be 
added  in  quite  notable  amounts  it  will  be  neutralized  by  the  acid 
product  of  the  fermentation,  and  under  such  circumstances  about 
all  that  has  been  accomplished  has  been  to  remove  one  of  the 
products  from  the  solution.  Strong  alkali  is  very  harmful  to  the 
ferment.  Green  found  in  the  first  work  upon  this  castor  bean 
ferment,  which  was  discovered  by  him,  that  acids  were  inimical ; 
hydrochloric  acid,  for  example,  in  fiftieth  normal  solution.  I 
have  never  tested  the  influence  of  this  acid,  but  acetic  acid  twen- 
tieth normal  is  not  inimical.  This  strength  of  acetic  acid  does 
not  of  course  contain  as  much  dissociated  hydrogen  as  50/N  HC1. 
It  is  furthermore  possible  that  the  action  is  due  to  the  molecule 
and  is  a  specific  chemical  influence  (sodium  chloride  is  also  de- 


VOL.  l]  Taylor. — On  Fermentation.  261 

scribed  as  harmful)  and  not  due  to  the  free  acidity.  It  would 
indeed  be  unusual  if  a  ferment  were  so  excessively  sensitive  to 
the  presence  of  its  natural  product  in  the  fermentative  reaction. 
The  powder  of  the  castor  bean  contains  over  1  per  cent,  of  ash, 
including-  both  sodium  and  potassium  chloride,  as  well  as  the  salts 
of  the  earthy  metals. 

An  interesting  instance  of  the  conservation  of  a  ferment  by 
the  presence  of  the  substance  related  to  it  as  the  substrate  is  to 
be  found  in  this  powder.  If  two  equal  masses  of  the  powder  be 
exposed  to  high  temperature  in  suspension  in  water,  and  to  the 
one  a  fat  be  added,  it  will  be  found  at  the  close  of  the  period  of 
heating  that  the  powder  containing  the  fat  will  have  resisted  de- 
struction by  heating  much  better  than  the  other  powder.  This 
fact  may  explain  why  the  ferment  is  stable  so  long  under  experi- 
mentation ;  it  may  be  due  to  the  fat  present,  since  these  are  lim- 
ited reactions  except  under  certain  conditions.  This  fact  may  be 
iissumed  to  be  the  result  of  a  combination  between  ferment  and 
substrate. 

Animal  and  vegetable  lipases  are  able  to  accelerate  the  cleav- 
age of  the  natural  fats,  of  the  esters  of  glycerine  with  the  lower 
members  of  the  fatty  acid  series,  of  the  esters  of  the  monatomic 
alcohols,  of  lecethins,  and  of  the  synthetic  asymetric  esters.  In 
all  probability  the  cleavage  of  hippuric  acid  and  of  salol,  and 
possibly  many  other  drugs,  in  the  intestinal  tract,  is  accomplished 
by  means  of  lipase. 

The  reaction  of  fat  fermentation.  The  reaction  of  the  cleav- 
age of  an  ester  is  an  act  of  simple  hydrolysis;  the  ester  adds 
water  and  is  then  divided  into  an  alcohol  and  a  fatty  acid.  Thus 
aethyl  acetate  -(-  water  =  aethyl  alcohol  -|-  acetic  acid.  The 
higher  fats  follow  the  same  type  of  reaction.  Euler  and  Gold- 
schmidt  have  attempted  to  interpret  the  reaction  from  the  point 
of  view  of  ionization.  Euler  found  that  the  equilibrium  of  the 
various  esters  in  moderately  dilute  solutions  was  not  constant; 
the  methyl  esters  were  hydrolyzed  the  least,  the  aethyl  esters  the 
most.  The  hydrolysis  of  an  ester,  he  determined  further,  was 
within  certain  limits  proportional  to  the  strength  of  its  acid,  that 
it  is  increased  with  the  dissociation  of  the  acid.  Upon  the  as- 
sumption that  the  ester  may  be  written  CHsCO — O — C2Hs  (using 


262  University  of  California  Publications.       [PATHOLOGY 

aethyl  acetate  for  an  illustration),  there  are  two  possibilities  for 
ionic  dissociation:  CH3CO+  —  OC2H5  and  CH3COO~  —  C2Hs". 
In  the  first  case  the  acid  would  act  as  the  base  (CHsCO — OH) 
and  the  alcohol  as  the  acid  (C^HoO — H) ;  in  the  second  case, 
the  alcohol  would  act  as  the  base  (C2H.  —  OH)  and  the  acid  as 
the  acid  (CH3000  —  H).  For  this  conception,  which  is  quite 
analogous  to  the  van't  Hoff  theory  of  saponification,  Euler  has 
adduced  some  experimental  evidence.  For  the  higher  fats  as  well 
as  for  the  simple  esters  it  is  known  that  an  autohydrolysis  occurs 
on  solution  in  water.  This  is  in  all  probability  due  to  the  disso-. 
ciated  hydrogen  ion  of  water.  Pure  fats,  free  of  bacteria,  ex- 
posed to  light  slowly  undergo  cleavage  into  the  glycerine  and 
fatty  acid.  Added  to  this  appears  next  an  auto-oxidation,  and 
thus  the  acids  found  following  such  an  auto-hydrolysis  are  fatty 
acids  and  oxyacids.  The  hydrolysis  is  greatly  accelerated  by 
heat,  and  hydrolysis  by  the  use  of  steam  is  the  common  com- 
mercial method  of  splitting  fats.  Hydrogen  ions  also  act  as 
great  accelerators,  and  colloidal  platinum  has  some  action. 
Whether  this  cleavage  occurs  through  the  medium  of  interme- 
diary reactions  is  not  known.  There  is  some  evidence  that  in 
the  cleavage  of  the  tri-glicerides  the  molecules  of  fatty  acid  are 
split  off  successively.  Lewkowitsch  has  shown  that  in  the  ordi- 
nary saponification  of  fats,  mono-  and  di-glycerides  may  be  iso- 
lated at  certain  stages  of  the  reaction.  In  the  synthesis  of  these 
fats  by  the  aid  of  heat  it  is  known  that  the  addition  of  the  first 
molecule  of  fatty  acid  may  be  accomplished  in  a  relative  mixture 
of  the  two  components  at  a  moderate  degree  of  heat.  For  the 
addition  of  the  second  molecule  of  fatty  acid,  an  excess  of  the 
fatty  acid  is  necessary,  and  a  much  higher  degree  of  heat  is  re- 
quired. For  the  addition  of  the  third  molecule  of  fatty  acid  the 
fatty  ^eid  is  no  longer  effective,  the  anhydride  must  be  employed 
in  excess,  and  the  reaction  is  then  greatly  accelerated  by  satura- 
tion of  the  system  with  an  anhydrous  salt  of  the  fatty  acid ;  and 
even  under  these  circumstances  a  still  higher  temperature  is  de- 
manded. The  act  of  cleavage  thus  seems  to  retrace  the  steps  of 
the  synthesis.  It  must  not,  however,  be  supposed  that  when,  in 
a  system  of  a  soluble  triglyceride  as  triacetin,  the  equilibrium  is 
established,  the  cleavage  be  one  to  the  diglyceride.  So  far  as 


VOL.  1]  Taylor. — On  Fermentation.  263 

the  reaction  extends,  it  is  a  complete  one  for  the  molecule;  the 
unsplit  fat  is  triacetin.  This  of  course  in  no  wise  represents  a 
contradiction  of  the  theory  of  the  step-like  hydrolysis ;  this  only 
states  that  the  cleavage  of  whatever  portion  of  the  fat  is  split 
occurs  in  successive  steps  corresponding  to  the  several  molecules 
of  fatty  acid. 

The  equilibrium  in  the  system  alcohol  -|-  acid  =  ester  -}- 
water  is  maintained  only  when  the  components  are  present  in 
appropriate  relations.  If  an  excess  of  the  alcohol  or  of  the  acid 
be  added,  all  of  the  mass  of  the  acid  or  alcohol,  as  the  case  may 
be,  will  be  combined  to  form  the  ester.  Thus  an  excess  of  alcohol 
will  lead  to  the  combination  of  all  the  acid,  while  upon  the  sim- 
ilar action  of  an  excess  of  the  acid  is  based  the  methods  for  the 
synthetic  preparation  of  these  fats.  On  the  other  hand,  if  an 
excess  of  water  be  present,  the  degree  of  hydrolysis  will  be  in- 
creased, and  if  the  system  be  sufficiently  diluted  with  water,  the 
ester  will  be  nearly  completely  split.  It  is  therefore  necessary 
before  employing  a  certain  concentration  of  ester  in  digestion 
experiments  to  determine  the  station  of  equilibrium  for  that  con- 
centration at  that  temperature.  This  may  be  best  accomplished 
by  means  of  an  appropriate  acid  hydrolysis,  it  being  assumed 
that  this  catalysor  does  not  alter  the  station  of  equilibrium.  I 
have  made  these  determinations  for  three  solutions  of  triacetin. 

The  solutions  were  made  acid  to  the  degree  of  1  mol.  sul- 
phuric acid,  and  the  ester  hydrolyzed  at  20°  for  several  months 
until  the  reaction  was  complete,  the  system  stationary.  For  a  2 
per  cent  solution  of  triacetin,  the  equilibrium  lies  at  ester  26  — 
products  74 ;  for  a  1  per  cent,  solution,  ester  18  —  products  82 ; 
for  a  one-half  per  cent,  solution,  ester  12  —  products  88  per  cent. 

Kinetics  of  lipolysis.  The  fermentation  of  fat  presents  an 
attractive  field  for  the  investigation  of  fermentation  from  the 
dynamic  point  of  view  because  the  hydrolysis  of  esters  has  been 
long  a  classical  subject  of  study,  representing  as  it  does  a  reac- 
tion of  definable  and  measurable  reversibility.  First  investigated 
by  Berthelot  and  St.  Gilles  in  1862,  the  reaction  was  recognized 
as  one  of  reversible  nature,  terminating  in  an  equilibrium  that 
was  approximately  the  same  irrespective  of  the  direction  from 
which  the  reaction  proceeded.  The  temperature  had  a  marked 


264  ruiversity  of  California  Publications.       [PATHOLOGY 

influence  upon  the  velocity  of  the  reaction,  and  some  influence 
upon  the  point  of  equilibrium.  For  the  different  fatty  acids  they 
found  that  the  velocity  of  reaction  diminished  with  increasing 
molecular  weight ;  the  same  rule  did  not  hold  for  different  alco- 
holds,  though  these  gave  widely  varying  though  irregular  results. 
Berthelot  considered  that  the  transformation  in  the  unit  of  time 
was  proportional  to  the  mass  of  the  reacting  bodies,  and  inversely 
proportional  to  the  volume.  In  their  experiments  they  deter- 
mined that  the  point  of  equilibrium  of  the  completed  reaction  lay 
at  two-thirds  mol.  ester  to  one-third  mol.  of  acid  and  alcohol ;  the 
reaction  could  be  reinaugurated  by  the  addition  of  either  ester  or 
alcohol  and  acid. 

The  present  formulation  of  the  reaction  is  somewhat  differ- 
ent. The  equation  previously  given  to  account  for  the  progress 
of  a  reversible  reaction  on  the  premises  of  the  law  of  mass  action 
is  known  not  to  hold  experimentally  for  the  reaction  now  under 
discussion,  because  it  assumes  that  the  combination  of  fatty  acid 
and  alcohol  in  the  one  direction  and  the  cleavage  of  the  ester  in 
the  other  direction  proceeds  at  a  rate  dependent  solely  upon  their 
several  masses,  and  upon  the  dissociated  hydrogen  of  the  solvent 
water;  this  is  not  true,  because  one  of  the  components,  acetic 
acid,  acts  as  an  auto-catalysor.  The  acetic  acid  is  in  part  disso- 
ciated, and  the  dissociated  acid  acts  as  a  positive  catalysor.  When 
alcohol  and  the  fatty  acid  are  mixed,  the  catalysor  is  present  in 
greatest  amount  at  a  time  when  the  active  masses  are  greatest, 
therefore  the  acceleration  in  the  direction  of  combination  is 
marked;  when  the  ester  and  water  are  mixed,  the  autocatalysor 
is  not  present,  and  appears  only  very  gradually,  is  greatest  when 
the  active  masses  are  small,  therefore  the  acceleration  is  not  pro- 
nounced in  the  direction  of  cleavage.  As  the  reaction  alcohol  -+- 
fatty  acid  proceeds  the  catalysor  is  gradually  reduced,  while  in 
the  other  reaction  it  is  gradually  increased.  When  the  station 
of  equilibrium  is  reached,  it  is  the  same  in  either  case,  but  it  re- 
quires a  longer  time  in  the  case  of  the  reaction  ester  -f-  water  than 
is  the  case  of  the  reaction  alcohol  -f-  fatty  acid.  Knoblauch  has 
shown  that  when  hydrochloric  acid  is  added  in  such  quantity  to 
each  system  as  to  make  negligible  the  influence  of  the  dissociated 
acetic  acid  (thus  maintaining  throughout  a  constancy  of  the  hy- 


TCL.  1]  Taylor. — On  Fermentation.  265 

•drogen  ions)  the  velocity  constants  of  the  opposing  reactions  are 
equal. 

Kastle  and  Loevenhart  have  studied  the  kinetics  of  the 
fermentation  of  ethyl  butyrate  by  animal  lipase.  They  found 
the  reaction  usually  incomplete ;  if  the  ferment  were  very  active 
or  the  substrate  concentration  very  low,  a  practically  complete 
reaction  was  sometimes  attained.  The  constants  of  velocity  di- 
minished through  the  course  of  an  experiment.  This  velocity 
was  not  directly  proportional  to  the  active  mass  of  the  substrate. 
With  constant  substrate  concentrations,  the  velocity  was  directly 
proportional  to  the  quantity  of  ferment.  They  found  the  opti- 
mal temperature  at  40°.  More  recently  Kastle  has  again  studied 
the  kinetic  relations.  He  found  that  the  animal  lipase  employed 
was  completely  retained  by  a  porcelain  filter.  He  demonstrated 
that  the  reaction  was  monomolecular,  and  that  in  general  the  re- 
sults followed  the  equation  for  a  reaction  of  that  order,  though 
there  was  some  lagging  which  was  attributed  to  the  acid  set  free. 
The  ferment  was  not  appreciably  destroyed  in  the  reaction.  The 
transformation  was  not  directly  proportional  to  the  substrate 
concentration.  He  also  determined  the  interesting  fact  that 
while  the  lipase  in  watery  solution  was  soon  destroyed,  it  would 
keep  for  a  long  time  in  a  very  weak  acid  solution. 

Volhard2  and  Stade,  working  with  gastric  lipase  and  yolk  of 
•egg,  found  the  velocity  of  transformation  proportional  to  the 
substrate  concentration.  Their  figures  are,  however,  only  rough , 
their  method  cannot  be  considered  accurate. 

There  are  theoretical  considerations  that  indicate  the  impos- 
sibility of  securing  quantitative  results  in  the  fermentation  of 
natural  fats,  and  these  are  based  on  the  inability  to  control  the 
substrate  concentration.  When  we  speak  of  a  fluid  fat,  we  mean 
a  fat  in  which  at  the  temperature  present  the  triolein  is  able  to 
hold  the  tripalmitine  and  tristearine  in  solution.  When  we  speak 
of  a  congealed  fat,  we  mean  a  fat  in  which  at  the  temperature 
present  the  triolein  is  not  able  to  hold  the  other  two  fats  in  solu- 
tion. The  triolein  is  a  solvent  for  the  other  two  fats.  The  so- 
called  melting  point  of  a  natural  fat  is  not  a  physical  melting 
point  at  all,  but  is  the  temperature  at  which  the  triolein  in  a  par- 
ticular fat  is  able  to  dissolve  the  tripalmitine  and  tristearine.  The 


266  University  of  California  Publications.       [PATHOLOGY 

oelic  acid  has  the  same  relations  of  solubility  for  both  the  pal- 
mitic and  stearic  acids  and  fats.  Now  in  a  fermentation  experi- 
ment, which  fat  is  first  attacked?  We  do  not  know,  though  there 
is  some  evidence  that  tristearine  is  most  easily  split.  But  which- 
ever were  first  hydrolyzed,  it  is  clear  that  the  substrate  concen- 
tration could  not  be  constant  through  the  experiment,  and  all 
the  more  so  when  it  is  realized  that  one  of  the  products  of  the 
reaction  (oleic  acid)  is  a  solvent  for  the  substrate.  The  relative 
insolubility  of  natural  fats  is  a  great  drawback.  If  one  were  to 
attempt  to  operate  with  fluid  fats  and  ferment  alone,  the  reac- 
tion (which  does  occur)  would  be  a  bimolecular  one,  and  for 
these  we  have  for  the  substances  and  circumstances  under  con- 
sideration no  mathematical  development. 

Since  one  ought  to  work  with  a  known  fat,  which  shall  it  be  ? 
The  lower  fats  of  the  saturated  series  are  soluble,  and  in  every 
way  adapted  to  the  needs  of  the  experiments.  As  a  rule  the  fats 
of  the  mon-atomic  alcohols  have  been  employed,  such  as  ethyl  bu- 
terate.  To  the  use  of  these  simple  esters  there  are  many  objec- 
tions. They  are  volatile,  and  the  substrate  concentration  is  hard 
to  control.  They  are  unstable.  They  exhibit  the  reversed  reac- 
tion (the  formation  of  the  ester  from  the  alcohol  and  acid)  to  a 
measurable  degree.  The  cleavage  is  measurably  accelerated  by 
acids,  and  thus  the  one  product  (the  fatty  acid)  acts  as  an  auto- 
catalysor.  Lastly  they  exhibit  a  much  greater  chemical  resistance 
to  ferments  than  do  the  esters  of  glycerine;  and  this  resistance 
is  in  marked  contrast  to  the  greater  readiness  of  their  hydrolysis 
by  acids,  to  which  the  glycerine  esters  are  highly  resistant. 

I  have  employed  in  my  studies  the  triglyceride  of  acetic  acid. 
This  may  be  prepared  upon  a  large  scale  in  a  state  of  almost 
absolute  purity,  free  of  water,  and  of  acidity.  It  is  scarcely  vo- 
latile, does  not  disintegrate,  the  auto-hydrolysis  does  not  occur 
with  measurable  rapidity  in  watery  solutions,  the  reversion  does 
not  occur  at  ordinary  temperatures  with  measurable  rapidity, 
the  substance  is  resistant  to  acids,  and  thus  auto-catalysis  by  the 
product,  acetic  acid,  is  slight.  To  illustrate  the  pronounced  char- 
acter of  these  qualities,  figures  may  be  given.  Adequate  fermen- 
tation tests  with  this  ester  may  be  completed  within  a  hundred 
hours,  at  room  temperature.  At  this  temperature  no  reversion 


TOL.  l]  Taylor. — On  Fermentation.  267 

of  the  reaction,  no  formation  of  ester  from  a  mixture  of  glacial 
acetic  acid  and  water-free  glycerine,  can  be  obtained  in  less  than 
a  couple  of  months.  In  the  concentration  of  substrate  employed, 
no  acidity  (auto-hydrolysis  of  the  ester)  will  appear  at  the  tem- 
perature employed  within  a  month.  The  addition  of  that  degree 
of  acidity  that  could  be  developed  at  the  completion  of  the  fer- 
mentation will  not  at  the  temperature  used  split  a  measurable 
amount  of  the  ester  within  a  week.  This  ester  is  very  susceptible 
of  fermentation,  particularly  with  the  lipase  of  the  castor  bean, 
with  which  I  have  most  experience.  These  qualities  make  work 
with  this  substrate  a  real  pleasure.  The  measurements  are  accu- 
rately accomplished  by  a  simple  titration.  As  against  the  higher 
members  of  the  same  series,  this  fat  has  the  great  advantage  of 
forming  a  true  homogeneous  solution. 

In  carrying  out  experiments  with  the  castor  bean  powder  it 
is  necessary  to  modify  somewhat  the  usual  method  of  procedure. 
In  an  ordinary  experiment  of  catalysos  in  a  homogeneous  system, 
and  in  such  fermentations  as  present  a  high  degree  of  intermix- 
ture, it  is  assumed  that  all  parts  of  the  mass  have  the  same  com- 
position. This  is  true  for  the  homogeneous  system ;  it  is  so  nearly 
true  for  an  ordinary  colloidal  mixture  that  the  error  may  be  neg- 
lected. But  in  systems  in  which  a  veritable  gross  suspension  and 
emulsion  exist,  such  an  assumption  may  no  longer  be  made.  It 
is  not  under  these  circumstances  permissible  to  prepare  a  large 
volume  of  the  system  under  investigation  and  from  time  to  time 
remove  a  portion  and  determine  the  degree  of  disappearance  of 
the  substrate  or  the  appearance  of  the  products.  One  is  com- 
pelled to  prepare  many  tests,  often  as  many  as  thirty  or  more, 
and  employ  one  for  each  quantitative  determination.  This  pro- 
cedure exposes  the  results  to  the  error  that  is  certain  to  follow 
the  slight  variations  in  the  preparation  of  the  individual  tests— 
the  errors  in  the  different  weighings,  in  the  titrations,  and  also 
the  error  in  the  assumption  that  each  gram  of  the  powder  has 
the  same  activity.  These  errors  may  be  minimized  by  a  very 
careful  mixing  of  the  powder,  accurate  weighing,  etc.,  but  they 
cannot  be  removed.  Proper  mixture  of  the  tests  means  also 
proper  and  continued  shaking.  This  is  less  important  in  fermen- 
tations of  soluble  than  of  suspended  fats.  A  lack  of  shaking  re- 


268  University  of  California  Publications.       [PATHOLOGY 

suits  in  a  retardation  of  the  reaction.  Fortunately  the  fat  reac- 
tions are  quite  slow,  so  that  the  factor  of  stirring  is  less  impera- 
tive than  in  very  rapid  reactions.  It  is  best  to  employ  a  mechan- 
ical shaking  apparatus. 

The  triacetin  was  prepared  according  to  the  method  of  See- 
gen,  and  was  rectified  twice  by  distillation  at  174°  at  25  mm. 
pressure.  It  was  colorless,  neutral,  and  entirely  soluble  in  fifty 
parts  of  water. 

The  temperature  varied  within  a  degree  of  18°  ;  we  had  no 
thermostat  large  enough  to  accommodate  the  numerous-  flasks. 
The  error  was  not  serious. 

Employing  triacetin  as  the  substrate,  I  have  found  that  the 
rate  of  transformation  follows  very  closely  the  logarythmal 
curve.  Using  solutions  of  one-half,  1  and  2  per  cent,  strength, 
with  one  gramme  of  ferment  in  the  vol.  of  100  c.c.,  at  18°,  the 
following  constants  were  obtained,  calculated  by  the  equation  K 

=  —  log  -   the  constants  are: 

t       "•  A—x 

t^hours.  4  8  16  24  28  32  40  48 


0.096   0.162   0.287   0.418   0.489   0.477   0.623   0.652 
A 

C    109    96     92     98     104     88     106    96 

1  %'   -—   0.083   0.174   0.338   0.418   0.488   0.542   0.609   0.655 

A 

C    94     104    112     98     104    106    102     96 

2  %"   —   0.098   0.174   0.323   0.431   0.502   0.485   0.595   0.636" 

A 

C    112    104    106    102    108     90     98     91 

Despite  the  fluctuations,  these  figures  are  quite  regular  when 
all  the  conditions  of  the  experiment  are  taken  into  consideration. 
They  may  be  justly  said  to  indicate  that  the  transformation  in 
the  unit  of  time  is  directly  proportional  to  the  active  mass  of  the 
substrate.  Apart  from  the  slight  irregularities,  these  figures  for 
the  constants  for  three  different  concentrations  of  substrate  are 
closely  enough  concordant  to  give  the  constants  a  real  value,  such 
a  value  as  they  should  have  according  to  the  equations.  This  sta- 
bility of  the  figures  for  the  constants  is  reinforced  by  another 


VOL.  l]  Taylor.  —  On  Fermentation.  269 

fact,  the  observation  that  the  same  stations  of  equilibrium  ob- 
served for  these  concentrations  with  the  use  of  sulphuric  acid  as 
the  catalysor,  are  closely  reproduced  by  these  fermentations. 

\%  \%  2% 

Acid  12/88  18—82  24—76 

Ferment    14—86  21—79  30—70 

This  result  shows  further  that  the  ferment  does  not  partici- 
pate in  the  reaction  in  any  other  capacity  than  as  a  catalysor, 
which  we  have  seen  is  not  true  of  other  ferments  as  a  rule. 

If    we    apply    to    these    results     the     equation     of     Henri 

—4^=(-  -  )  it  follows  that  m  and  n  are  equal. 

dt      .     l  +  m(A—  Jc]  - 


Nicloux  investigated  the  cleavage  of  olive  oil  by  the  castor 
bean  enzyme.  He  found  that  when  the  ferment  was  present  in 
considerable  amount,  the  constants  calculated  for  the  reaction  in 
a  monomolecular  reaction  were  quite  constant. 

Stade  has  studied  the  fermentation  of  the  fat  in  the  yolk  of 
egg  by  gastric  lipase.  The  constants,  as  calculated  from  his  re- 
sults by  Euler,  display  a  rapid  and  progressive  diminution,  which 
was  ascribed  to  the  influence  of  the  fatty  acid. 

Other  studies  have  been  carried  out  upon  emulsified  fats  by 
Fromme,  Engel,  Henri  and  Nicloux,  Zellner,  and  Kanitz.  Nicloux 
and  Henri  and  Nicloux  found  the  transformation  roughly  pro- 
portional to  the  time,  though  with  a  higher  concentration  of  sub- 
strate there  was  some  relation  to  the  substrate  mass.  Zellner 
found  the  transformation  constant  in  the  unit  of  time,  while 
Kanitz  found  the  transformation  roughly  proportional  to  the 
square  root  of  the  time.  The  experiments  of  Volhard,  Stade, 
Fromme,  Engel,  as  well  as  of  the  earlier  experiments  of  Conn- 
stein,  Hover,  and  Warteburg,  were  not  carried  out  from  the  point 
of  view  of  the  law  of  mass  action,  and  a  recalculation  of  their 
data  gives  irregular  results. 

I  have  further  studied  the  hydrolysis  of  aethyl  acetate,  and 
here  the  curious  observation  was  made  that  this  ester  is  much 
more  resistant  to  this  ferment  than  is  triacetin.  For  ordinary 
acid  hydrolysis  or  saponification.  aethyl  acetate  is  more  easily 
hydrolyzed  than  triacetin.  but  with  this  ferment  the  relations 
are  reversed.  Not  only  is  aethyl  acetate  resistant  to  the  ferment, 


270  University  of  California  Publications.       [PATHOLOGY 

but  the  results  are  so  irregular  as  to  be  of  little  value.  This  rela- 
tion to  these  two  esters  is  also  true  of  pancreatic  lipase ;  it  digests 
triacetin  much  more  easily  than  aethyl  acetate. 

Very  interesting  have  been  the  results  with  triolein.  The 
triolein  was  prepared  by  fractional  saponification  of  olive  oil  in 
the  cold,  extraction  with  aether,  washing  with  sodium  carbonate, 
and  purified  by  several  times  being  passed  through  acetone  and 
aether.  It  was  neutral,  of  a  pale  yellow  color,  odorless,  and  en- 
tirely soluble  in  water-free  aether.  It  gave  the  iodine  number  85, 
the  saponification  number  191.  This  was  employed  in  2  per  cent, 
suspension,  with  1  per  cent,  suspension  of  the  ferment  powder. 
The  results  for  the  observations  is  as  follows ;  t  is  days. 

c.c.  10/  normal  acid,  tempt.  18.0. 

123457  9  11  16          18 

3       5       7.5       9       11       16       21.6       27.2       32.1       38.1 

These  give  the  following  increases  in  acidity  for  each  day. 

3  —  2  —  2.5  —  1.5  —  2  —  2.5  —  2.5  —  2.8  —  2.8  —  2.8  - 
2.8  —  2.2  —  2.2  —  2.2  —  2.7  —  3.7  - 

Though  it  must  be  stated  that  there  is  quite  an  error  in  the 
method  of  estimation — an  extraction  with  aether  followed  by  the 
titration  of  the  aether  alcoholic  solution  of  the  oleic  acid— it  is 
apparent  that  the  rate  of  transformation  is  a  function  of  the 
time.  A  somewhat  similar  result  has  been  reported  by  Henri 
and  Nicloux.  They  found  that  for  the  ferment  of  the  castor  bean 
there  was  an  optimum  concentration  of  acid.  When  at  the  larg- 
est optimum  concentration  of  acid  they  fermented  different  con- 
centrations of  oil,  varying  from  25  to  87  units,  the  transforma- 
tions in  all  were  approximately  identical  in  the  unit  of  time. 
Their  ferment  was  much  stronger  than  any  employed  by  me. 

The  system  triolein-lipase-water  is  a  two-phase  system  in  the 
strict  sense  of  the  term.  The  lipase  and  triolein  are  almost  in- 
soluble in  water.  It  may  in  all  probability  be  assumed  that  the 
triolein  and  the  lipase  are  in  a  colloidal  complex;  the  minute 
traces  in  solution  may  be  assumed  to  be  in  a  complex  chemical 
combination.  When  the  first  fractions  of  the  triolein  have  been 
hydrolyzed,  the  one  product,  glycerine,  passes  into  the  water 
phase,  in  all  probability  quite  completely.  The  other  product, 
oleic  acid,  may  be  assumed  to  be  divided  between  the  water  phase 


VOL.  l]  Taylor. — On  Fermentation.  271 

and  the  colloidal  phase,  in  large  part  in  the  latter.  That  frac- 
tion of  the  oleic  acid  present  in  the  water  will  render  the  triolein 
more  soluble  than  in  water  alone.  The  reaction  may  be  assumed 
to  occur  on  the  boundary  film  of  the  colloidal  complex.  As  the 
reaction  proceeds,  the  dimension  of  this  boundary  film  is  dimin- 
ished. Under  these  circumstances,  the  experimental  finding — 
that  the  transformation  is  a  linear  function  of  time — is  best  ex- 
plained by  the  Xernst  hypothesis  that  the  experimental  velocity 
is  a  diffusion  velocity.  That  the  experimental  findings  do  not 
correspond  well  with  the  theory  of  reaction  in  a  homogeneous 
system  is  indicated  also  by  a  further  consideration  of  the  condi- 
tions in  the  experiment. 

Triolein,  glyceride  of  oleinic  acid,  is  almost  insoluble  in 
water;  it  is  so  little  soluble  that  a  saturated  solution  at  ordi- 
nary temperature  could  not  be  employed  for  even  a  qualita- 
tive study.  Oleic  acid  is  very  insoluble;  it  is  so  insoluble  and 
so  little  dissociated  that  a  saturated  solution  in  water  at  ordi- 
nary temperature  scarcely  conducts  the  current  better  than  the 
water  with  which  the  solution  has  been  prepared.  But  the  satu- 
rated solution  of  oleic  acid  is  a  much  better  solvent  for  the 
triolein  than  is  the  water;  and  analogously  a  solution  satu- 
rated with  triolein  and  oleic  acid  is  the  best  watery  solvent  for 
the  other  higher  fats.  Now,  discarding  for  the  moment  the  solv- 
ent action  of  the  fatty  acid,  an  experiment  with  the  hydrolysis 
of  triolein  would  always  present  a  saturated  solution  of  the  sub- 
strate ;  if  one  began  with  sufficient  triolein  to  actually  accomplish 
an  experiment  of  value,  one  would  begin  with  a  suspension,  and 
as  in  each  moment  a  certain  amount  of  the  ester  would  be  hydro- 
lyzed,  its  place  in  the  solution  would  be  taken  by  a  new  portion 
that  had  passed  into  solution.  Therefore  the  substrate  concen- 
tration would  through  the  greater  part  of  the  experiment  until 
almost  the  end  remain  constant,  and  according  to  the  law  of  mass 
action,  the  transformation  in  the  unit  of  time  would  remain  con- 
stant. Since  the  solubility  of  the  one  product,  oleic  acid,  is  also 
very  low,  this  would  soon  begin  to  be  thrown  out  of  solution. 
Thus  we  would  have  the  condition  of  a  catalytic  reaction  in  a 
system  saturated  with  the  substrate  and  saturated  with  one  of 
the  products — a  most  anomalous  condition.  This  is  what  actually 


272  University  of  California  Publications.       [PATHOLOGY 

occurs  in  an  acid  catalysis  of  a  fat ;  in  each  moment  fatty  acid  is 
being  thrown  out  of  solution,  and  in  each  corresponding  moment 
a  fraction  of  fat  is  dissolving ;  and  whereas  in  the  beginning  there 
was  a  heterogeneity  in  the  system  due  to  the  substrate  (the  fat), 
at  the  close  of  the  reaction  there  remains  a  .heterogeneity  in  the 
system  due  to  the  one  product,  the  fatty  acid.  Under  these  cir- 
cumstances, the  reaction  becomes  practically  a  complete  cleavage, 
and  does  not  display  a  point  of  equilibrium  such  as  is  usually 
seen  in  the  hydrolysis  of  an  ester.  In  the  film  of  contact  between 
the  phase  of  the  solvent  and  of  the  suspended  fat,  the  substrate 
is  in  hypersaturated  solution ;  and  as  the  reaction  there  occurs 
with  rapidity,  there  must  exist  in  this  film  also  supersaturation 
of  the  fatty  acid.  On  stirring  the  mixture,  as  this  fatty  acid 
diffuses  from  the  film  of  contact  it  will  pass  out  of  solution.  The 
process  continuing  in  this  manner,  the  reaction  would  proceed 
until  the  fat  had  all  passed  into  solution,  and  the  velocity  of  the 
reaction  would  depend  solely  upon  the  rate  of  diffusion.  These 
reactions  would  be  in  part  altered  by  the  effect  of  the  oleic  acid 
upon  the  solubility  of  the  fat,  since  in  this  behavior  the  oleic  acid 
plays  the  role  of  a  solvent,  and  thus  alters  the  concentration  of 
the  substrate  and  increases  its  active  mass.  This  in  itself  might 
be  of  little  importance,  since  the  reaction  in  the  film  may  be  so 
rapid  as  to  be  independent  of  the  substrate  concentration  in  the 
sense  of  the  law  of  mass  action.  But  it  might  be  of  importance 
if  the  rate  of  diffusion  of  the  bodies  were  altered  by  the  presence 
of  the  greater  content  of  fat  and  fatty  acid  in  the  solvent.  In 
all  likelihood,  the  rate  of  diffusion  is  somewhat  altered ;  but  this 
is  probably  of  no  practical  account,  because  the  rate  of  diffusion 
of  fats  and  fatty  acids  of  low  solubilities  is  exceedingly  slow.  It 
is,  however,  probable  that  in  this  case  the  increase  of  the  sub- 
strate brought  about  by  the  fatty  acid  might  lead  to  an  apprec- 
iable increase  in  the  velocity  of  reaction  at  the  film  of  contact, 
because  the  unaided  solubility  of  the  fat  would  be  so  extremely 
low.  In  any  event,  it  is  not  probable  that  the  alterations  effected 
by  the  oleic  acid  would  determine  notable  quantitative  distur- 
bances, because  though  it  is  assumed  in  the  Nernst  theory  that 
the  substrate  must  be  so  slightly  soluble  that  it  does  not  partici- 
pate appreciably  in  the  diffusion,  even  with  the  aid  of  the  oleic. 


VoL- l]  Taylor.— On  Fermentation.  273 

acid,  the  solubility  of  the  fat  is  so  slight  as  to  lie  well  within  this 
reservation.  I  cannot  see  how  the  Xernst  theory  does  not  in  every 
way  correspond  to  the  conditions  in  an  acid  hydrolysis  of  a 
higher  fat.  In  the  earlier  consideration  of  this  theory  I  stated 
that  for  many  fermentations  the  heterogeneity  of  the  system  was 
often  so  slight  that  it  was  doubtful  whether  the  conditions  of  the 
theory  were  adequately  fulfilled:  but  in  the  case  of  the  higher 
natural  fats  the  system  is  typically  heterogeneous,  as  typically 
so  as  in  the  reactions  studied  by  Brunner  under  the  direction  of 
Nernst.  It  is  true  that  the  reaction  velocity  is  a  function  of  time 
in  a  reaction  in  a  homogeneous  system  where  the  active  substrate 
is  constant,  the  condition  postulated  above  for  this  system.  But 
since  the  application  of  this  point  of  view  to  reactions  in  a  het- 
erogeneous system  make  the  velocity  also  a  function  of  the  dimen- 
sions of  the  boundary  of  contact  of  the  two  phases,  the  theory 
would  not  fit  the  facts  here  because  while  the  transformation  is 
uniform  the  dimensions  are  constantly  decreasing.  The  oleic 
acid  is  passing  out  of  solution,  it  is  true,  but  the  reactions  cannot 
be  supposed  to  occur  at  the  boundary  of  its  particles. 

The  substrate  is  a  suspended  particle,  very  slightly  soluble  in 
the  medium  (though  this  solubility  will  be  somewhat  increased 
by  the  presence  of  one  of  the  products),  and  this  substrate  dif- 
fuses very  slowly;  the  products  are  very  slightly  soluble  in  the 
medium  (the  fatty  acid  at  least:  the  alcohol  is  soluble)  and  dif- 
fuses very  slowly ;  and  the  catalysor  is  a  suspended  particle,  and 
the  reaction  may  be  assumed  to  occur  in  large  part  or  wholly  in 
the  film  of  contact  of  the  catalysor  and  substrate.  Obviously 
there  are  two  streams  of  diffusion:  the  one  connected  with  the 
solution  of  the  substrate,  and  the  second  connected  with  the  reac- 
tion at  the  film  of  contact  of  the  solution  and  the  catalysor.  It 
seems  certain  that  when  one  considers  the  facts — considers  the 
prolonged  constancy  of  the  active  mass  of  the  substrate  from  the 
point  of  view  of  the  law  of  mass  action,  the  known  rapidity  of 
the  reaction  in  the  film  of  contact  of  two  phases,  the  demon- 
strable slowness  of  the  diffusion,  and  finally  the  necessity  of  two* 
diffusions,  one  must  believe  that  here  certainly  the  theory  of 
Xernst  does  apply  with  full  force,  and  that  in  accordance  there- 
with the  so-called  reaction  velocity  of  a  fat  fermentation  with 


274  University  of  California  Publications.       [PATHOLOGY 

the  use  of  fats  of  the  higher  series  in  reality  represents  a  diffu- 
sion velocity  solely.  As  Heimbrodt  has  shown,  under  proper  con- 
ditions of  concentration,  the  formulation  of  the  theory  leads  to 
an  equation  quite  identical  with  that  corresponding  to  a  mono- 
molecyjar  reaction  in  a  homogeneous  system. 

This  interpretation  of  the  phenomenon  is  supported  also  by 
the  observations  on  the  effect  of  increase  in  temperature  on  the 
fermentation.  An  increase  of  10°  does  not  double  the  velocity; 
it  increases  it  not  over  one-fourth,  just  such  an  increment  as 
would  be  expected  from  the  known  influence  of  increase  of  tem- 
perature on  diffusion  velocity.  Senter,  in  a  recent  paper,  states 
that  the  same  relation  to  temperature  holds  for  the  reduction  of 
hydrogen  peroxide  by  hamase,  the  blood  peroxidase,  the  increase 
of  velocity  with  increase  in  temperature,  were  not  what  would  be 
expected  in  a  chemical  reaction,  but  corresponded  well  to  what 
would  be  expected  in  a  diffusion  experiment. 

The  point  of  view  of  Herzog  cannot  be  applied  to  this  diges- 
tion at  all  because  the  viscosity  of  the  system  is  practically  un- 
changed throughout  the  experiment.  The  emulsion  triolein-fer- 
ment  powder-water  has  the  same  viscosity  as  the  emulsion  oleinic 
acid-glycerine-ferment  powder-water,  within  the  range  of  con- 
centrations at  least  that  are  involved  in  these  experiments. 

Relation  of  acceleration  to  mass  of  ferment.  The  acceleration 
of  the  hydrolysis  of  triacetin  has  been  found  d,irectly  propor- 
tional to  the  ferment  present.  This  is  illustrated  in  the  following 
experiment. 

Substrate  constant:  (a)  ferment  2  per  cent;  (&)  ferment  1 
per  cent.  The  times  are  those  necessary  for  the  proportional 
transformation  of  the  stated  units  of  acid. 

units  10         20          30 

a     time        23         51         104 
6     time        47        97         216 

Kastle  and  Loevenhart,  working  with  animal  lipase  and 
aethyl  butyrate  and  monobutyrin,  found  the  acceleration  pro- 
portional to  the  mass  of  ferment.  Vollhard  and  Stade,  working 
with  gastric  lipase  and  the  yolk  of  egg,  found  the  acceleration 
roughly  proportional  to  the  square  root  of  the  ferment.  It  is 
clear,  however,  that  the  yolk  of  egg  method  cannot  be  used  for 


VoL-  !]  Taylor. — On  Fermentation.  275 

quantitative  study.  Engel  also  found  the  Schutz  rule  to  hold 
for  lipase,  and  this  has  been  stated  further  by  members  of  the 
Pawlow  school,  and  also  by  Benach  and  Gayot.  Engel  compared 
the  times  necessary  to  split  off  1  per  cent,  of  acid;  the  Pawlow 
procedure,  on  the  contrary,  is  to  compare  the  work  done  in  a  fixed 
time — a  theoretically  improper  procedure. 

The  reversion  of  the  fermentation.  This  has  been  repeatedly 
accomplished,  first  by  Kastle  and  Loevenhart  and  by  Bernizone 
for  aethyl  buterate,  by  Hanriot2  for  monobutryin,  both  employing 
animal  lipase.  In  a  previous  publication  I  reported  upon  the 
synthesis  of  triolein  through  the  activity  of  the  castor  bean  fer- 
ment, and  upon  the  failure  of  the  synthesis  of  the  same  fat  by 
pancreatic  lipase,  the  latter  experience  being  but  the  repetition 
of  the  previous  failures  of  other  investigators. 

Pottevin  has  recently  described  the  synthesis  of  numerous 
esters,  including  those  of  oleinic  and  stearic  acid,  by  pancreatic 
powders,  operating  in  the  absence  of  water.  The  report  states 
that  the  water-free  fatty  acids  are  suspended  in  the  alcohol  and 
the  dry  powder  added,  and  that  this  powder  does  not  go  into 
solution.  Theoretically  the  synthesis  ought  to  occur  with  rapid- 
ity, since  it  must  be  assumed  to  occur  at  the  boundary  of  contact 
of  the  ferment  phase.  The  conclusion  of  the  author,  that  the 
pancreatic  lipase  acts  as  a  hydrolytic  ferment  in  watery  solution, 
and  as  a  combining  ferment  in  the  absence  of  water,  is  not  sup- 
ported by  the  experimental  evidence,  and  may  be  rejected  on 
theoretical  grounds.  If  enzymic  fat  synthesis  occur  at  all  in  nat- 
ural life,  it  occurs  always  in  the  presence  of  water.  I  have,  how- 
ever, been  able  to  confirm  the  Pottevin  statement  for  triolein. 

Theoretically  the  acetic  acid  would  act  as  an  auto-catalysor, 
but  practically  no  such  action  is  to  be  discerned  in  the  constants. 
It  might  be  supposed  that  possibly  an  auto-catalysis  just  covered 
the  loss  of  the  ferment,  and  thus  hid  itself;  but  direct  experi- 
ments to  this  end  indicate  that  no  material  inactivation  of  the 
ferment  occurs  in  an  experiment  of  such  length.  The  equilib- 
rium may  be  brought  about  by  the  addition  of  the  theoretical 
amount  of  acetic  acid  (the  alcohol  has  little  effect)  ;  the  neutral- 
ization of  the  acetic  acid  will  reestablish  the  reaction  in  a  system 
in  equilibrium,  as  will  the  addition  of  more  substrate.  The  addi- 


276  University  of  California  Publications.       [PATHOLOGY 

tion  of  more  ferment,  however,  will  not  reinaugurate  the  reaction 
in  a  system  in  equilibrium.  If  one  mix  the  acid  alcohol  and  ester 
in  the  proportions  determined  to  constitute  the  equilibrium,  the 
addition  of  ferment  will  not  result  in  any  alteration  of  these  re- 
lations. 

Conditions  of  maximum  activity.  For  animal  lipase  the  state- 
ments concerning  the  favorable  reaction  are  contradictory.  A 
faint  alkalinity  is  stated  by  some  to  be  most  favorable,  by  others 
a  faint  acidity.  Obviously  a  faint  alkalinity  soon  means  neu- 
trality in  the  experiment,  since  the  fatty  acid  set  free  in  the  re- 
action- would  neutralize  the  reaction.  Animal  lipase  is  so  sensi- 
tive and  is  in  such  a  mixture  of  unstable  substances  that  it  seems 
to  vary  widely  with  each  different  preparation.  In  the  body  it 
acts  in  a  neutral  reaction.  In  a  test  with  the  higher  fats,  the 
products  of  the  reaction  could  never  give  rise  to  a  marked  acidity. 
The  vegetable  lipases  are  very  stable  substances  under  favorable 
circumstances.  Alkali  is  deleterious,  the  fatty  acids  not  at  all  so 
in  moderate  concentrations.  The  heavy  metal  salts  kill  the  castor 
been  lipase  rapidly,  but  the  ordinary  electrolytic  salts  (the  chlo- 
rides and  sulphates  of  potassium,  sodium  magnesium,  and  cal- 
cium) have  no  effect  at  low  concentrations.  Vegetable  lipase  is 
resistant  to  digestion,  particularly  to  pepsin.  The  results  with 
trypsin  are  complicated  by  the  vulnerability  to  alkali.  The  pow- 
dered castor  bean  contains  a  proteolytic  ferment,  which,  however, 
seems  less  capable  of  attacking  it  than  does  mammalian  tryp- 
sin. Purification  of  this  ferment  is  a  waste  of  time ;  the  fat-free 
powdered  beans  give  the  best  results  so  far  as  the  integrity  of 
the  ferment  is  concerned.  Of  zymoexciters  I  have  found  none. 
Bile  and  lecethin,  that  are  zymoexciters  of  animal  lipase,  do  not 
act  so  for  the  castor  bean  lipase,  rather  the  contrary.  Extracts 
of  liver  or  other  tissue  do  not  enhance  the  action  of  this  lipase, 
nor  do  extracts  of  the  leaves  and  stalks  of  the  castor  plant. 

According  to  Hewlitt,  lecethin  acts  as  a  powerful  zymoex- 
citer  to  pancreatic  lipase.  It  is  without  action  on  vegetable 
lipase.  Magnus  has  described  a  zymoexciter  that  exists  in  the 
liver;  it  is  a  diffusible  substance,  is  not  soluble  in  aether,  and  is 
not  destroyed  by  boiling.  According  to  Hoyer,  mangano  salts 
act  as  zymoexciters  to  vegetable  lipase. 


VOL.  1]  Taylor— On  Fermentation.  277 

Chemical  properties  of  ferment.  Lipase  has  not  been  isolated 
in  even  the  rough  form  that  has  been  accomplished  for  other 
ferments.  It  is  retained  to  some  extent  by  filtration  through 
paper,  to  a  greater  extent  by  filtration  through  infusorial  and 
porcelain  filters ;  it  has  no  power  of  dyalisis.  The  ferment  seems 
quite  insoluble  in  the  true  sense ;  the  suspensions  are  always  very 
colloidal.  All  preparations  that  I  have  seen  gave  typical  reac- 
tions for  protein.  The  suggestion  of  Hanriot  that  lipase  might 
be  an  iron-organic  complex  is  devoid  of  chemical  support.  On 
suspension  in  water  in  the  absence  of  an  ester,  lipase  is  rather 
rapidly  destroyed.  This  destruction  seems  to  be  a  simple  hy- 
drolysis, and  the  velocity  is  in  general  proportional  to  the  mass 
and  increases  with  increase  in  temperature.  The  products  of  the 
fermentation  of  the  natural  facts  act  in  a  protective  manner,  al- 
though to  less  extent  than  do  the  fats.  The  esters  of  the  common 
alcohols  do  not  protect  lipase  from  hydrolysis  as  do  glycerine 
fats,  while  the  products  are  directly  injurious.  This  action  is  the 
property  of  the  alcohol;  lipase  is  sensitive  to  aethyl  alcohol  in 
low  concentration. 


278  University  of  California  Publications.       [PATHOLOGY 


LITERATURE. 

Volhard.1     Zeitschr.  f.  klin.  Med.,  43,  397. 
Benech  and  Guyot.     C.  r.  Soc.  Biol.,  55,  719,  721. 
Hanriot.1    C.  r.  Soc.  Biol.,  48,  925. 

C.  r.  Acad.  Sc.,  123,  753. 
Euler.     Zeitschr.  f.  physik.  Chem.,  36,  405. 
Knoblauch.     Zeitschr.  f.  physik.  Chem.,  22,  268. 
Kastle  and  Loevenhart.     Am.  Chem.  Jour.,  24,  491. 
Kastle,  Johnson  and  Elove.     Am.  Chem.  Jour.,  31,  521. 
Volhard.2    Zeitschr.  f.  klin.  Med.,  42,  414;  43,  397. 
Volhard.    Zeitschr.  f.  klin.  Med.,  42,  414;  43,  397. 
Stade.     Beitr.  z.  chem.  Physiol.  u.  Path.,  3,  291. 
Seelig.     Berichte,  24,  3466. 
Nicloux.     C.  r.  Soc.  biol.,  56,  840. 
Fromme.     Beitr.  z.  chem.  Physiol.  u.  Path.,  7,  51. 
Engel.     Beitr.  z.  chem.  Physiol.  u.  Path.,  7,  77. 
Henri  and  Nicloux.     C.  r.  Soc.  Biol.,  57,  175. 
Zellner.     Monatsch.  f.  Chem.,  26,  727. 
Kanitz.     Zritschr.  f.  physiol.  Chem.,  46,  482. 
Connstein,  Hoyer,  and  Warteburg.    Berichte,  35,  3988. 
Heimbrodt.     Ann.  d.  Physik.  (IV),  13,  1028. 

Bernizone.    Atti  del.  Soc.  Lig.  di.  Scien.  nat.  e  Geograf.  Genoa,  //, 
Hanriot.-    C.  r.  Soc.  biol.,  53,  70, 
Pottevin.    C.  r.  Acad.  Sc.,  138,  378. 
Taylor.     Univ.  Cal.  Pub.  Pathology,  1,  33. 
Hewlitt.     Jour.  Med.  Ees.,  11,  377. 


V(ir-  1]  Taylor. — 0??  Fermentation.  279 


THE  NATURE  OF  FERMENTS. 
THE  REACTION  OF  FERMENTATIONS. 

We  know  very  little  of  the  chemical  nature  of  ferments. 
They  are  always  associated  with  numerous  bodies  derived  from 
the  cells  to  which  they  owe  their  origin,  and  they  are  so  labile 
that  isolation  is  attended  with  denaturation  and  decomposition. 
We  may  say  that  ferments  are  proteins,  or  closely  resemble  them. 
This  general  conclusion  is  based  upon  the  facts  that  ferments  are 
usually  coagulable,  they  respond  to  the  color  tests  and  reactions 
for  protein,  they  are  precipitated  by  the  ordinary  salts,  they 
yield  on  hydrolysis  or  digestion,  to  which  they  are  all  more  or 
less  susceptible,  amido  acids.  These  observances  are,  however, 
alike  in  the  case  of  no  two  ferments.  Their  physical  properties 
likewise  vary.  Some  are  very  colloidal,  others  diffuse.  Some 
rotate  light,  and  are  thus  known  to  contain  asymmetrical  carbon ; 
others  are  optically  inactive.  Some  contain  a  carbohydrate 
moiety,  and  thus  appear  to  resemble  glycoproteids.  Others  con- 
tain a  large  amount  of  phosphorus  in  organic  combination,  and 
on  digestion  yield  purin  bodies,  and  thus  appear  to  resemble  neu- 
cleoproteids.  In  fact  the  best  studied  of  the  animal  ferments, 
pepsin,  exhibits  these  qualities.  Other  ferments  contain  no  neu- 
clein  or  carbohydrate,  are  not  coagulable,  and  resemble  proteose. 
Thus  only  the  most  tentative  opinions  may  be  passed  upon  the 
chemical  nature  of  these  bodies.  Loew  and  his  students,  despair- 
ing of  a  general  characterization  of  the  nature  of  ferments,  have 
attempted  to  define  the  active  groups  of  the  molecule.  They  as- 
sume that  ferments  are  able  to  accelerate  the  velocity  of  reac- 
tions by  the  possession  of  active  labile  groups.  And  these  Loew 
considers  to  be  in  all  probability  keton  and  amino  groups.  These 
conclusions  are  based  upon  the  experimental  study  of  such  chem- 
ical procedures  whose  reactions  may  be  assumed  to  indicate  such 
groups;  thus  the  action  of  hydrazine,  cyanogens,  and  methyla- 
mine  indicate  the  .presence  of  keton  groups;  the  presence  of 
amino  groups  is  indicated  by  the  reactions  Avith  nitrous  acid  and 


280  University  of  California  Publications.       [PATHOLOGY 

dicyanogen.  It  is  doubtful  whether  these  considerations  are  of 
general  application.  Pollok  has  apparently  shown  that  by  the 
action  of  acid  and  alkali  on  trypsine  the  ferment  may  be  robbed 
of  its  power  of  digesting  albumine  and  globuline  and  fibrin,  with- 
out any  loss  in  the  faculty  of  digesting  gelatine.  Without  as- 
suming that  trypsine  is  a  mixture  of  ferments,  each  more  or  less 
specific  for  particular  proteins,  he  suggests  that  these  different 
functions  are  associated  with  different  group  radicals  of  the 
molecule. 

The  chemical  nature  of  fermentation.  A  discussion  of  the 
chemical  nature  of  the  fermentative  acceleration  must  in  the 
nature  of  the  phenomenon  be  based  primarily  upon  a  study  of 
the  nature  of  catalytic  accelerations  in  general,  and  upon  the 
demonstration  of  analogies  and  reactional  relations  existing  be- 
tween them.  Since  it  seems  certain  from  the  thermodynamic 
point  of  view  that  a  catalysor  or  ferment  acts  only  by  the  direct 
or  indirect  reduction  of  the  internal  resistance,  and  not  by  any 
increase  in  the  driving  force  of  the  reaction,  all  investigations 
must  be  directed  to  the  internal  physical  and  chemical  resistance. 

In  the  consideration  of  the  modus  operandi  of  the  catalytic 
acceleration,  we  thus  face  directly  the  question  of  the  nature  of 
the  internal  resistance  to  chemical  reactions  that  is  a  property  of 
all  substances.  Since  a  catalytic  acceleration  is  defined  as  an 
acceleration  due  to  the  lowering  of  the  internal  resistance  of  the 
substance,  we  must  attempt  to  define  a  conception  of  chemical 
resistance.  There  has  been  little  study  of  this  aspect  of  the  ques- 
tion. Of  the  two  factors  in  every  reaction — the  driving  force  and 
the  internal  resistance — nearly  all  the  physico-chemical  research 
has  been  directed  to  the  driving  force.  That  the  constitution  of 
organic  molecules  is  associated  with  variations  in  the  resistance 
to  reactions  is  of  course  well  known.  Examples  of  such  relations 
will  be  given  in  the  chapter  on  specificity  of  ferment  action.  But 
the  same  factor  of  resistance  resides  in  the  most  simple  inorganic 
substance.  In  any  event,  it  is  certain  that  the  term  internal  re- 
sistance stands  not  for  one  thing,  but  may  stand  for  many  things 
that  are  varied  from  case  to  case.  Under  these  circumstances, 
therefore,  the  means  whereby  a  catalytic  agent  lowers  this  resist- 
ance may  vary  from  case  to  case,  and  must  be  studied  anew  for 


VOL.  1]  Taylor. — On  Fermentation.  281 

e;ich  individual  reaction.  One  of  the  future  achievements  in 
chemistry  will  be  to  define  an  Ohm's  law  for  chemical  reactions. 

There  are  several  ways  in  which  the  presence  of  a  catalysor 
might  accelerate  the  velocity  of  a  reaction.  Firstly  it  might  re- 
duce the  chemical  resistance  of  a  substance,  just  as  temperature 
does:  it  might  operate  against  some  of  the  inhibitory  influences 
of  which  van 't  Iloff  speaks.  Secondly,  it  might  reduce  the  num- 
ber of  intermediate  reactions  that  occur  naturally  in  the  reaction. 
An  illustration  of  this  in  the  domain  of  inorganic  chemistry  is 
to  be  seen  in  the  action  of  a  cobalt  salt  on  the  reaction  between 
XaOH  and  Cl.  This  would  amount  to  a  short  cut  to  the  final 
product,  and  if  the  velocity  of  each  step  were  no  greater  than 
in  the  ordinary  reaction,  the  total  velocity  would  be  greater. 
Thirdly,  new  intermediary  reactions  might  be  introduced,  of 
greater  velocity,  so  that  the  sum  of  their  velocities  would  be 
greater  than  the -velocity  of  the  original  reaction.  A  quantita- 
tive illustration  of  this  is  to  be  found  in  the  acceleration  of  the 
reduction  of  hydrogen  peroxide  by  hydriodic  acid  under  the  ca- 
talytic influence  of  molybdic  acid. 

The  theory  of  intermediary  reactions  is  not  only  the  oldest 
theory,  having  been  suggested  by  Clement  and  Desormes,  Mits- 
cherlich,  Traube,  Schoenbein,  and  de  la  Rive,  but  it  has  in  its 
favor  a  large  amount  of  experimental  evidence  by  Engler, 
Woehler,  Wagner,  Haber,  Erode,  Kastle  and  Loevenhart,  Luther, 
Schilow,  Bach  and  Cordat,  and  others.  The  theory  is  in  brief 
that  a  catalysor  accelerates  the  velocity  of  a  reaction  by  the  in- 
troduction of  intermediary  reactions  with  the  formation  of  un- 
stable products,  usually  of  the  type  of  addition-products;  that 
these  products  are  themselves  so  unstable  that  they  disintegrate 
of  their  own  accord  or  they  are  disintegrated  by  the  action  of 
other  bodies  in  the  system ;  and  that  the  sum  of  the  velocities  of 
the  several  reactions  is  greater  than  the  velocity  of  the  primary 
reaction.  Wegscheider1  further  separates  simple  accelerations 
(Folgewirkungen)  from  accessory  accelerations  (Nebenwirkun- 
gen),  depending  upon  whether  the  end-product  of  the  acceler- 
ated reaction  is  the  same  as  in  the  unaccelerated  reaction.  The 
theory  has  been  tested  solely  upon  catalyses  of  pure  reactions, 
usually  of  inorganic  nature.  Thus  far  the  evidence  is  almost 


282  University  of  California  Publications.       [PATHOLOGY 

entirely  qualitative  ;  in  the  work  of  Erode  upon  the  acceleration 
of  the  reaction  between  hydrogen  peroxide  and  hydriodic  acid 
by  molybdic  acid,  the  kinetic  relations  were  worked  out  in  such 
a  manner  as  to  demonstrate  that  the  sum  of  the  velocities  of  the 
several  reactions  was  greater  than  the  velocity  of  the  natural  re- 
action. 

It  is  usually  assumed  that  even  the  simplest  reactions  are 
not  accomplished  directly;  they,  too,  pass  through  intermediary 
stages  (Wegscheider)  ;  and  thus  the  catalysor  by  the  introduc- 
tion of  other  intermediary  stages  does  not  in  the  least  alter  the 
general  nature  of  the  process  of  reaction,  but  by  introducing  in- 
termediary reactions  with  less  of  chemical  resistance  effects  a 
short  cut  to  the  stage  of  equilibrium.  Experimental  researches 
indicate  that  while  in  perhaps  the  majority  of  these  accelerations 
the  number  of  intermediary  reactions  is  increased,  instances  are 
known  in  which  the  number  of  intermediary  reactions  has  been 
diminished.  In  any  event,  be  the  number  in  the  catalytic  series 
greater  or  less,  the  theory  assumes  that  the  sum  of  their  velocities 
is  greater  than  was  the  velocity  of  the  original  reaction.  Ob- 
viously tlie  theory  can  be  properly  tested  only  upon  reaction  for 
which  we  can  determine  the  intermediary  reactions  with  and 
without  the  catalysor.  These  intermediary  reactions  are  to  be 
studied  from  the  standpoint  of  the  Ostwald  law  of  reaction- 
stages  :  In  all  chemical  reactions  the  most  stable  condition  is  not 
Attained  at  once,  but  either  the  nearest  reaction  is  attained  or 
among  several  possible  reactions  the  most  unstable,  etc.,  etc. 
Thus  Cu  SO4  =  2  KOH  =  K2S04  +CuO  +  H20  passes  through 
one  intermediary  stage. 

CuS04  +  2  KOH  =  K2S04  +  Cu(OH)2. 
Cu(OH)2  =  CuO  +  H20. 


Now  when  to  such  a  system  an  appropriate  positive  catalysor 
is  added,  new  intermediary  stages  are  introduced,  termed  for  a 
certain  large  class  of  reactions  stages  of  primary  oxides  ;  the  pri- 
mary oxides  have  some  of  the  behaviors  of  peroxides;  they  are 
stronger  oxidizors  than  the  highest  stable  oxidation-stage  and 
stronger  reducers  than  the  lowest  oxidation-stage  (Luther). 

2  H2O2  =  2  H2O  +  O2. 


VOL.  l]  Taylor.  —  On  Fermentation.  283 

In  the  presence  of  ferric  oxide  this  reaction  is  very  rapid. 

*2  H2O,  +  Fe2O3  =  iron  primary  oxide   (Fe2O3.O2)  +2  H,O. 
Fe2O3.O2  =  Fe»O3  +  O2. 

A  reaction  in  more  stages  is  as  follows  : 

4  NaOH  +  2  CL  =  4  NaCl  +  2  H2O  +  O.. 

L'4  NaOH  +  12  CL  =  12  H,O  +  12  NaCl  +  12  NaClO. 
\:>  NaClO  =  8  NaCl  +  4  NaClO,. 
4  NaClO3  =  NaCl  +  3  NaClCv 
3  NaClO4  =  3  NaCl  +  6  O2. 

Here  there  are  three  stages  of  primary  oxides,  and  each  more 
"primary"  than  the  succeeding:  one.  When  a  cobalt  salt  is  added 
the  reaction  is  accelerated. 

24  NaOH  +  12  CL  =  12  NaCl  +  12  H2O  +  12  NaClO. 

12  NaClO  +  cobalt  salt  =  12  NaCl  +  cobalt  primary  oxide. 

Cobalt  primary  oxide  =  cobalt  salt  +  6  O2. 

Here  all  the  NaCl-  are  formed  in  the  first  and  second  stages  ;  that 
is,  the  number  of  intermediary  reactions  is  reduced. 

The  reaction  of  potassium  permanganate  with  hydrochloric 
acid  has  its  direct  expression  in  the  following  formula  : 
KMnO4  +  8  HC1  —  KC1  +  MnCL  +  4  H2O  +  5  Cl. 

Platinic  chloride  accelerates  this  reaction  in  the  following  way  : 
KMnO4  +  4  H2PtCl6  =  KCl  +  MnCL  -f  4  PtCl4  +  4  H2O  +  5  Cl. 


the  platinic  chloride  having  combined  with  the  hydrochloric  acid 
to  form  the  chloroplatinum  acid  H,PtCl6,  which  is  more  rapidly 
reacted  upon  by  permanganate  than  is  hydrochloric  acid. 

The  reaction  between  hydrogen  peroxide  and  hydriodic  acid 
is  expressed  in  the  following  equation  : 
H2O2  +"2  HI  =  2  H2O  +  I2- 


01-  +  2H+  +  I-  -  H20  +  I2. 
When  molybdic  acid  is  added  to  the  system  we  have,  expressed 

in  its  simplest  terms  : 

H2O,,  +  H2MO4  =  H^10,;. 

H4MO«  +  2  HI  =  H2M04  +  2  H2O  +  I.. 

The  sum  of  the  velocities  of  these  reactions  is  very  much  greater 
than  in  the  original  reaction.     (Erode.) 


284  University  of  California  Publications.       [PATHOLOGY 

Kastle  and  Loevenhart  have  recently  discussed  the  action  of 
hydrogen  peroxide  and  have  given  strong  evidence  for  the  older 
view  that  whenever  hydrogen  peroxide  acts  as  an  oxidizing  or 
reducing  agent  it  first  combines  with  the  substance  to  form  an 
unstable  peroxide-like  body. 

Thus        H2S03  +  H202  =  H4SOr,. 

H4SO5  =  H,SO4  +  H,O. 
And        Ag20  +  3  H202  =  H4Ag2O6  +  H2O. 

H4Ag2O6  =  2  Ag  +  2  H2O  +  2  O,. 

Closely  related  to  the  theory  of  intermediary  reaction  is  the 
Kessler  theory  of  induction.  When  two  reactions  are  going  on 
in  the  same  solution,  the  presence  of  one  may  accelerate  the  ve- 
locity of  the  other.  On  close  analysis  it  is  seen  that  the  actual 
process  is  one  of  intermediary  reaction.  All  instances  of  reac- 
tion by  induction  are  of  course  not  catalytic,  but  many  of  them 
cannot  be  otherwise  defined  on  account  of  the  existence  of  a  slow 
primary  reaction.  Theoretically,  induced  reactions  may  be  di- 
vided into  two  groups :  those  in  which  the  intermediary  products 
are  stable,  and  those  in  which  they  are  labile.  In  catalyses  we 
have  apparently  to  deal  with  those  in  which  the  intermediary  re- 
actions are  labile.  The  two  bodies  in  the  reaction  whose  presence 
results  in  the  acceleration  (the  primary,  voluntary  reaction)  are 
termed  the  inductor  and  the  actor,  while  the  body  whose  reaction 
is  induced  to  an  acceleration  is  termed  the  acceptor,  the  actor 


being  the  same  in  each  reaction.  The  intermediary  body  may  be 
either  a  combination  of  the  actor  with  the  inductor,  or  it  may  be 
an  unstable  addition  product  of  the  actor  or  of  the  inductor. 
Good  illustrations  may  be  given  from  the  group,  of  oxidations,, 
though  the  phenomenon  is  not  at  all  confined  to  oxidations;  it  is, 
oifthe  contrary,  in  all  probability  a  phenomenon  of  widespread 
occurrence  and  fundamental  importance.  The  simplest  type  is 
where  the  acceptor  D  is  slowly  reacting  with  the  actor  A;  when 
the  inductor  7  is  added  it  also  reacts  with  the  actor  A,  and  then 
the  product  reacts  with  the  acceptor  Z>,  as  a  result  of  which  the 
actor  A  associated  with  7  is  transferred  to  7>;  that  is,  7  induces', 
more  of  A  to  react  with  D  than  before. 


VOL.  1]  Taylor. — On  Fermentation.  285 

D  +  A  =  DA  —  very  slow. 

1  +  A  =  IA  —  very  rapid ;  then 
IA  +  D  =  I  +  DA, 

so  that  the  velocity  of  the  formation  of  DA  is  increased. 

The  reaction  SO2  +  O  =  S03  is  very  slow.  When  in  the  sys- 
tem, however,  the  reaction  ferrous  salt  -f-  oxygen  =  ferric  salt 
is  going  on,  the  combination  of  the  sulphur  dioxide  is  greatly 
accelerated.  The  reaction  between  the  iron  salts  acts  as  the  car- 
rier of  oxygen ;  as  fast  as  the  ferric  oxide  is  formed  it  is  reduced 
by  the  sulphur  dioxide. 

SO2  +  O  =  SO3  —  slow. 

2  FeO  +  O,  =  2  FeO2  (possible  Fe2O3)  rapid;  then 
So,  +  FeO,  =  SO3  +  FeO. 

The  reaction  from  the  ferrous  to  the  ferric  salt  must  be  kept 
going  by  an  appropriate  catalysor.  This  method  now  constitutes 
a  commercial  method  for  the  manufacture  of  sulphuric  acid. 
Many  of  the  induced  reactions  are  not  so  simple,  in  that  there  is 
no  reaction  between  the  product  of  the  second  reaction  and  the 
acceptor.  For  example,  chromic  acid,  or  its  salts,  is  not  able  to 
oxidize  tartaric  acid  with  measurable  velocity;  it  oxidizes  arsen- 
ious  oxide  with  great  rapidity.  When  the  two  reactions  are  ac- 
complished in  the  same  system  the  tartaric  acid  is  also  oxidized. 

Chromic    acid  +  tartaric   acid  =  formic    acid   and    other   acids  — 

very  slow. 

Chromic  acid  +  arsenious  acid  =  arsenic  acid  —  very  rapid. 
Chromic  acid  -f-  tartaric  acid  =  formic  acid  —  rapid. 

Here  the  product  of  the  primary  reaction,  arsenic  acid,  is  stable ; 
when  the  reaction  is  completed  the  arsenious  acid  is  entirely  oxi- 
dized, the  tartaric  acid  in  large  part.  The  relations  have  there- 
fore not  been  those  of  an  oxygen  carrier,  as  in  the  first  illustra- 
tion. The  explanation  is  that  some  intermediary  stage  in  the 
reaction  chromic  acid  -j-  arsenious  acid  provides  the  point  of  de- 
parture for  the  impetus  of  the  second  reaction.  This  may  be 
represented  as  follows : 

Chromic    acid  +  arsenious   acid  =  intermediary   product  =  arsenie 

acid. 
Intermediary  product  +  tartaric   acid  =  formic   acid,   etc. 

Another  illustration  is  furnished  by  bromic  acid,  which  does  not 
act  upon  arsenious  acid,  but  reacts  rapidly  with  sulphurous  acid :. 


286  University  of  California  Publications.       [PATHOLOGY 

when  the  reactions  are  associated,  the  arsenious  acid  is  also  oxi- 
dized. 

As  the  subject  of  inductions  is  investigated,  it  becomes  ap- 
parent that  many  of  the  accelerations  by  intermediary  reactions 
are  of  this  nature.  All  the  activations  of  oxygen,  in  which  the 
formation  of  peroxide-like  bodies  is  probable,  belong  to  the  sim- 
pler reactions  by  induction.  Indeed  the  coupled  reactions  should 
be  a  sub-class  of  the  transformations  by  intermediary  reactions. 
It  will,  on  the  contrary,  not  be  possible  to  class  all  the  catalyses 
as  induced  reaction,  numerous  and  important  as  these  certainly 
are.  Luther  and  Schilow  have  recently  studied  these  inductions, 
and  believe  that  the  process  follows  one  of  two  relations,  depend- 
ing upon  whether  the  intermediary  stage  is  stabile  or  labile.  And 
of  the  latter,  the  intermediary  stage  that  acts  as  the  accelator  to 
the  induced  reaction  may  be  either  a  combination  of  the  actor 
with  the  inductor,  or  a  higher  oxidation  stage  of  the  actor  or  the 
inductor. 

The  oldest  known  theory  of  intermediary  reaction  is  that  de- 
vised to  explain  the  acceleration  exerted  by  the  oxides  of  nitrogen 
upon  the  oxidation  of  sulphur  dioxide  in  the  manufacture  of  sul- 
phuric acid.  The  recent  studies  of  Trautz  have  apparently  dem- 
onstrated that  there  are  more  intermediary  reactions  than  had 
been  previously  assumed.  The  sum  reaction  may  be  represented 
in  the  reaction  :  2  NO  +  0  +  H20  +  SO,  =  2  NO  -f  H..SO,. 
Trautz  was  able  to  determine  experimental  evidence  for  the  fol- 
lowing reactions : 

NO  +  NO2  +  H2O  =  2  NO.OH. 

S03H 

->  (2  NO.SOaH  +  2  H2.)  ->  NO  -f  NO  +  2  H2O. 

SO,H 

->  3  NO  +  2  H2S04. 


2  NO  +  O  +  2  H2SO4  —  2  NO2.SO3H  +  H2O. 

2  ONO.S03H  +  2  H20  +  NO(SO3H)2—  3  NO  +  4  H2SO4. 

ONO  SO;,H  xn  OTT    ,    TT  on 

,     jj    JJQ  —  l  +  JiabLV 

2  NO  +  O2  =  2  NO2. 


VOL.  1]  Taylor. — On  Fermentation.  287 

The  catalysor  NO  acts  to  a  certain  extent  as  an  anti-catalysor. 
The  distinction  from  the  coupled  reactions  is  apparent.  The  most 
interesting  phase  for  us  lies  in  the  fact  that  the  catalysor  NO  re- 
acts not  only  with  the  oxygen  and  the  substrate  S02,  but  also  with 
the  product  S0:!,  and  that  from  the  reactions  between  the  product 
and  some  of  the  higher  reaction-stages  a  portion  of  the  velocity  of 
the  process  is  derived.  This  is  of  importance  in  furnishing  an 
inorganic  illustration  to  what  is  undoubtedly  of  frequent,  pos- 
sibly of  regular  occurrence,  the  reaction  of  combination  between 
the  ferment  and  the  product  of  its  acceleration. 

A  closely  analogous  condition  in  the  organic  work  seems  to 
lie  in  the  phenomenon  of  the  action  of  acids  upon  the  formation 
of  isomers  of  cinchonin  as  described  by  Skraup.  When  cinchonin 
is  exposed  to  the  action  of  hydrochloric  acid  (or  other  halogens), 
one  isomeric  base  is  produced,  the  a-i-cinchonin,  and  the  HC1 
addition-product  of  cinchonin. 

/HC1 — cinchonine  (addition  reaction) 
Cinchonine  -}-  HC1  =  / 

a — i — cinchonine  (transformation  reaction) 

Secondary  reaction:      a — i — cinchonine  +  HC1  =  HC1 — a — i — cinchonine. 

The  first  idea  would  naturally  be  that  suggested  by  Wislescenus 
for  the  transformation  in  such  reactions,  that  the  addition-pro- 
duct represented  the  intermediary  product.  Skraup,  however, 
has  shown  that  the  addition  product  with  HC1  is  not  to  be  con- 
verted into  HC1  and  the  isomeric  base  under  the  conditions  of 
the  experiment.  The  reaction  has  since  been  considered  from 
the  kinetic  point  of  view  by  Wegscheider,-  who  interprets  the  re- 
lations as  follows.  When  HC1  and  cinchonin  are  brought  into  a 
system  two  reactions  occur,  probably  in  definite  proportions  and 
in  accordance  with  the  law  of  mass  action ;  the  end  products  of 
the  two  reactions  are  firstly  the  ITC1  addition  product  of  cincho- 
nin. and  secondly  the  isomeric  base,  a-i-cinchonin.  The  first  re- 
action or  product  acts  in  some  way  as  the  catalysor  for  the  second 
reaction,  the  transformation  into  the  isomeric  base.  It  is  certain 
that  the  concentration  of  the  hydrogen  ions  does  not  determine 
the  velocity  of  the  formation  of  the  isomeric  base.  The  acceler- 
ating influence  of  the  side  reaction  of -addition  upon  the  reaction 
of  transformation  may  be  regarded  as  one  of  two  procedures. 


288  University  of  California  Publications.       [PATHOLOGY 

Either  some  intermediary  form  of  the  addition-reaction  consti- 
tutes an  intermediary  form  of  the  transformation-reaction;  i.e., 
in  the  series  of  intermediary  forms  of  the  addition-reaction  is  a 
point  where  the  process  may  go  on  to  the  reaction  of  transforma- 
tion, a  point  where  the  line  of  least  chemical  resistance  lies  in  the 
direction  of  the  transformation-reaction;  or  the  two  lines  of 
direction  are  early  separated  and  some  product  of  the  addition- 
reaction  acts  as  a  catalysor  to  the  transformation-reaction,  and 
produces  with  this  reaction  intermediary  forms  that  carry  with 
them  a  heightened  velocity  of  this  second  reaction.  Wegscheide,r 
inclines  to  the  view  that  the  first  reaction  alone,  the  addition- 
reaction,  is  an  auto-reaction ;  he  believes  that  the  second  reaction 
is  not  one  that  exists  per  se  and  is  simply  accelerated  by  the  first 
reaction,  but  that  the  first  reaction  or  its  products  actually  calls 
the  second  reaction  into  being. 

We  meet  here  with  an  apparent  contradiction  of  the  state- 
ment that  a  fermentation  is  an  acceleration  of  an  already  exist- 
ing reaction.  If  in  a  catalysis  or  fermentation  the  end  product  is 
different  than  that  yielded  in  the  unaccelerated  reaction,  the  re- 
lations suggest  a  reaction  de  novo.  When  the  relations  are  care- 
fully scrutinized,  however,  it  seems  clear  that  we  are  dealing  not 
with  a  contradiction,  but  with  an  extension  of  the  principle, 
van't  Hoff  has  pointed  out  that  in  some  catalytic  reactions  the 
factor  of  internal  chemical  resistance  has  been  such  as  to  inhibit 
the  unaccelerated  reaction ;  but  he  does  not  term  such  a  catalytic 
reaction  a  reaction  de  novo.  In  a  similar  manner,  even  though 
in  some  instances  the  end  product  be  different  in  the  accelerated 
and  the  unaccelerated  reaction,  it  is  the  existence  of  the  primary 
unaccelerated  reaction  that  makes  possible  the  secondary  reac- 
tions in  the  process  of  acceleration  that  yields  the  end  end  pro- 
ducts. In  the  domain  of  organic  substances  the  liability  is  so 
great  and  the  possibilities  of  reactions  so  numerous  that  many 
possibilities  for  the  installation  of  side  reactions  are  presented  in 
the  catalyses  and  fermentations  of  such  substances.  This  may  be 
illustrated  in  the  following  scheme : 

Substrate  4-  water  =  pu  =r  Pb  =  PC  =  end  product  A.      (auto-reaction). 

Substrate  +  water  +  ferment 

=  pr  =  pa  =  pt  —  pu  =  end  product  A.     (accelerated  reaction). 
-   ])y  =  pz  =  end  product  B.      (side  reaction). 


1]  Taylor.— On  Fermentation.  289 

Obviously  the  side  reaction  is  not  a  reaction  de  novo,  but  is 
as  essentially  an  acceleration  (and  deviation)  of  an  auto-reaction 
as  is  the  accelerated  reaction  that  yields  the  same  end  product  as 
the  auto-reaction.  And  were  the  entire  trend  of  the  reaction  to 
take  the  side  path  and  product  B  appear  as  the  sole  end  product, 
that  fact  would  hold  just  as  true.  In  many  of  the  cases  we  are 
dealing  with  uncompleted  or  superimposed  reactions.  Thus  sugar 
may  apparently  be  fermented  to  alcohol  and  to  acetic  and  lactic 
acid.  Xow  there  can  be  little  doubt  that  the  lactic  acid  fermen- 
tation consists  in  the  reaction  as  described  for  alcoholic  fermen- 
tation checked  at  the  stage  of  lactic  acid;  and  the  acetic  acid 
fermentation  is  an  oxidation  fermentation  of  alcohol.  Up  to  the 
present  therefore  we  have  no  data  tending  to  indicate  that  fer- 
mentations are  ever  reactions  de  novo. 

An  interesting  form  of  catalysis,  and  one  doubtless  of  wide- 
spread occurrence-  in  the  inorganic  world,  consists  in  the  literal 
formation  of  a  galvanic  cell  within  the  reacting  system,  the  ca- 
talysor  not  participating  in  the  chemical  reaction  directly.  A 
good  illustration  is  seen  in  the  action  of  copper  upon  the  solution 
of  metallic  zinc  by  sulphuric  acid.  The  reaction  with  the  pure 
substances  follows  the  following  equation : 

H2S04=  2  H+  +  SOj. 
Zn-f  2  H+  =  Zn 


This  reaction  is  slow,  as  is  familiar  to  every  one  who  has  at- 
tempted the  preparation  of  hydrogen  from  the  pure  reagents.  A 
trace  of  copper  will  accelerate  it  greatly,  by  the  formation  of  a 
galvanic  cell  with  a  closed  circuit. 

Anode  Zn.      Zn  =  Zn++ +  2  U. 

Cathode.      H2S°4  = 
Cu 

In  closed  circuit:      2  U  +  2  U  =  O. 

The  reaction  is  very  rapid,  ana  is  doubtless  illustrative  of  fre- 
quent disintegrations  of  impure  metals. 


290 


University  of  California  Publications.       [PATHOLOGY 


For  organic  reactions  intermediary  products  have  been  less 
often  demonstrated,  on  account  of  the  greater  complexity  of  the 
relations.  Some  are,  however,  known.  The  first  demonstrated 
instance  (which  is  now  known  to  be  susceptible  of  marked  ca- 
talytic acceleration)  was  made  by  Williamson  for  the  formation 
of  aether  from  alcohol  through  the  action  of  sulphuric  acid, 
aether-sulphuric  acid  being  shown  to  represent  the  intermediary 
stage. 

H2SO4  +  C2H3.OH  =  (02H5)H  SO4.  +  H2O. 
(C2H3)  H  SO4  +  C2H5.OH=:(C2H5)20  +  H2SO4. 

When  propyl  alcohol  is  heated  with  sulphuric  acid,  a  mole- 
cule of  water  is  withdrawn ;  thereupon  another  molecule  of  water 
is  added,  though  in  a  different  way,  so  that  isophopyl  alcohol  is 
formed. 

CH3.CH2.CH2OH  —  H2O  =  CH3.CH  :CH2  (propylen) . 
CH3.CH  :CH2  +  H2O  =  CH3.CHOH.CH3. 

The  formation  of  acroline  from  glycerine  by  heating  is  illus- 
trated in  the  following  series : 


CH2.OH] 

I 
CH  OH  j-  —2  HoO= 

I 
CH2.OHJ 

glycerine 


fCH,         ] 

!      I 

\  v 

\  I 

I  CH  .OH 


fCH2             1 

f  CH2 

1 

|    | 

-4  CH                 —  H2' 

0=^  CH 

1    1 

1    1 

L  CH.(OH2)  J 

L  CH.O 

acrolein  hydrate 

acrolein 

According  to  the  generally  accepted  theory,  when  water  and 
carbon  dioxide  are  in  contact  in  the  presence  of  sunlight  formal- 
dehyde is  slowly  formed,  and  the  acceleration  of  this  reaction  is 
supposed  to  constitute  the  first  step  in  the  assimilation  of  carbon 
by  plants.  The  reaction  may  be  regarded  as  passing  through  the 
intermediary  stage  of  formic  acid. 

H,O  +  CO2  =  H.COOH  +O. 
H.COOH  =  H.COH  +O. 

An  interesting  catalytic  intramolecular  transformation  de- 
scribed years  ago  by  Zincke  and  Kuester  affords  another  good 
illustration.  The  ketone  of  C5C100  presents  several  isomers,  and 
twro  of  these  in  particular  tend  always  to  pass  into  each  other 
and  to  establish  an  equilibrium  in  the  mass.  The  reaction  is  ap- 
parently not  direct,  and  although  the  intermediary  body  has  not 


VOL.  1] 


Taylor. — On  Fermentation. 


291 


been  isolated,  the  evidence  seems  to  indicate  that  the  reaction 
follows  the  following  equation  : 


Cl  C 


C1C 


C  C12        C12C 


C  C12        C12C 


C  C12       Cl  C 


C  Cl, 


C12C 


CO 


CC1 


CC12 


An  additional  illustration  is  furnished  in  the  equations  for 
the  fermentation  of  d-glucose  to  alcohol  and  carbon  dioxide,  given 
in  the  lecture  on  alcoholic  fermentation.  There  the  one  inter- 
mediary product  is  lactic  acid,  which  has  been  confirmed,  while 
the  first  is  still  unknown,  though  a  glyoxylic  body  seems  recently 
to  have  been  identified  in  the  intermediary  series.  These  ex- 
amples could  be  multiplied  by  illustrations  from  the  literature. 
The  sole  reason  why  the  general  theory  of  the  occurrence  of  inter- 
mediary stages  in  all  reactions  cannot  be  confirmed  in  each  con- 
crete instance  lies  in  the  instability  of  these  products  and  in  the 
transitoriness  of  their  appearance. 

Apart  from  the  considerations  adduced  for  alcoholic  fermen- 
tation (and  for  the  oxydases  and  peroxydases)  there  have  been 
few  studies  of  fermentations  from  the  point  of  view  of  interme- 
diary reactions.  As  a  rule  the  conditions  are  so  complex  and 
uncontrollable  that  we  do  well  if  we  are  able  to  estimate  the 
march  of  the  reaction  and  the  nature  of  the  final  products,  with- 
out even  attempting  the  isolation  of  intermediary  reactions.  In 
the  fermentations  of  the  hexoses,  in  the  platinic  accelerations  of 
cleavages  of  carbohydrates,  and  in  the  reactions  of  hydrogen 
peroxide  with  organic  bodies  the  relations  promise  soon  to  be 
sufficiently  clear  to  permit  of  investigations  from  this  point  of 
view.  We  know  that  ferments  are  very  labile  bodies,  that  pre- 
cisely such  as  labile  bodies  have  the  tendency  to  enter  into  combi- 
nations of  high  lability,  and  we  may  infer  that  in  this  very  qual- 
ity lies  their  adaptability  to  the  acceleration  of  slow  reactions. 
But  this  very  lability  makes  the  intermediary  products  so  very 
elusive  and  incapable  of  isolation,  so  that  qualitative  results  are 
probably  all  that  may  be  hoped  for  in  the  near  future.  Schoen- 
bein  spoke  of  the  reactions  of  hydrogen  peroxide  as  the  "Urbild 


292  University  of  California  Publications.       [PATHOLOGY 

aller  Gaehrung, ' '  and  this  terse  sentence  is  yearly  becoming  more 
impressive.  There  was  a  time  when  the  reactions  of  hydrogen 
peroxide  were  as  little  understood  as  are  those  of  the  common 
ferments  to-day,  and  it  is  not  too  much  to  hope  that  as  much  pro- 
gress may  be  made  with  the  latter  within  the  next  decade  as  has 
been  made  during  the  last  three  decades  upon  the  study  of  the 
catalyses  with  hydrogen  peroxide.  To  this  end,  however,  we 
shall  need  to  prepare  ferments  in  a  much  greater  state  of  purity 
than  is  now  possible,  in  order  that  secondary  and  extraneous  re- 
actions shall  be  excluded  from  the  system. 

Although  we  are  not  as  yet  able  in  concrete  instances  of  fer- 
mentation, apart  from  alcoholic  fermentation,  to  point  out  the 
intermediary  reactions,  we  h&ve  an  indirect  argument  for  this 
theory  in  the  fact  that  the  ferment  is  known  to  combine  with  the 
substrate.  While  it  is  true  in  the  general  sense  that  the  action 
of  catalysors  is  peculiar  in  this,  that  there  is  no  stochiometric 
relation  between  the  catalysor  and  the  ferment,  it  is,  on  the  other 
hand,  equally  true  that  on  the  theory  of  intermediary  reactions, 
during  the  moment  of  reaction  there  must  be  a  stochiometric  re- 
lation between  them.  The  statement  that  there  is  no  stoechio- 
metric  relation  between  the  catalysor  and  the  reaction  it  accel- 
erates is  true  only  in  the  relative  sense  that  there  is  no  stochio- 
metric relation  between  the  mass  of  the  catalysor  and  the  mass  of 
initial  substrate  or  the  final  products.  But  so  long  as  we  locate 
the  modus  operandi  of  catalytic  acceleration  in  intermediary  re- 
actions, there  must  obviously  be  a  stochiometric  relation  between 
the  catalysor  and  the  substrate  in  the  moment  of  reaction.  This 
is  as  true  of  colloidal  platinum  as  it  is  of  ferrous  sulphate.  It  is 
the  rapidity  of  the  intermediary  reactions,  the  putting-cm  and 
casting-off  of  the  reaction,  so  to  speak,  that  gives  the  gross  ap- 
pearance of  absence  of  a  stochiometric  relation.  Only  on  such  a 
basis  can  the  relation  of  degree  of  acceleration  to  mass  of  cataly- 
sor or  ferment  be  explained.  It  is  my  conviction  that  when  the 
common  fermentations,  the  reactions  of  monomolecular  order,  are 
carefully  studied,  it  will  be  found  that  in  all  the  degree  of  accel- 
eration is  proportional  to  the  mass  of  the  ferment.  This  is  just 
what  we  should  expect,  since  the  only  relations  in  the  reaction  are 
the  masses  of  the  substrate  and  the  ferment.  It  might  be  assumed 


VOL-  !]  Taylor. — On  Fermentation.  293 

that  the  molecules  of  ferment  /  each  combined  with  one  of  sub- 
strate s.  This  would  give  us  /  -f  s  =  fs  =  intermediary  product 
(one  or  more)  =end  product  -f  f.  This  f  would  then  combine 
with  another  molecule  of  substrate,  and  the  process  be  repeated. 
On  the  basis  of  this  scheme,  the  degree  of  acceleration  would 
naturally  be  proportional  to  the  number  of  /,  at  least  within  cer- 
tain limits  of  relations  of  concentration. 

There  have  been  several  other  considerations  urged  to  explain 
the  accelerations  of  catalysis  and  fermentation.  These  theories 
do  not  exclude  the  proposition  of  intermediary  reactions.  Euler 
has  urged  the  application  of  the  theory  of  ionization  to  this  entire 
group  of  reactions.  His  hypothesis,  which,  though  unsupported 
by  much  experimental  evidence,  is  founded  upon  solid  general 
considerations,  regards  the  action  of  the  catalysor  as  a  positive 
influence  on  the  magnitude  of  ionization  of  the  reacting  bodies. 
The  presence  of  the  reacting  body  is  considered  to  so  alter  the 
concentration  of  the  active  ions  that  the  reaction  is  hastened. 
"Chemical  catalyses  depend  upon  alterations  in  the  concentra- 
tions of  one  or  more  of  the  molecules  that  carry  on  the  unaccel- 
erated  reaction,  i.e.  (by  the  application  of  the  electro-chemical 
principle  to  the  general  field  of  chemistry),  upon  an  increase  (or 
a  decrease)  of  the  ions  that  participate  in  the  reaction." 

The  hypothesis  of  Bodenstein  rests  upon  the  proposition  that 
there  exists  about  the  catalysor  a  zone  of  increased  concentration 
through  which  the  velocity  of  the  reaction  would  be  hastened. 
This  hypothesis  is  obviously  most  applicable  to  catalysis  within  a 
heterogeneous  system. 

The  validity  of  the  general  principles  underlying  these  two 
hypotheses  cannot  be  denied.  At  the  same  time,  it  cannot  be  be- 
lieved to-day  that  they  alone  can  explain  a  catalysis  or  a  fermen- 
tation, independent  of  the  existence  of  intermediary  reactions. 

The  work  of  Bredig  and  his  pupils  on  the  catalytic  action  of 
colloidal  suspensions  of  metals  has  tended  to  exaggerate  the  phys- 
ical aspect  of  the  subject.  Granting  unreservedly  the  accessory 
influences  that  the  physical  properties  of  colloidal  suspensions 
(their  enormous  surface  tension,  etc.)  may  possess  upon  a  react- 
ing system,  the  fact  remains  that  the  catalytic  influence  of  col- 
loidal metals  must,  too,  be  attributed  to  intermediary  reactions. 


294  University  of  California  Publications.       [PATHOLOGY 

The  colloidal  state  must  be  held  to  endow  the  metal  with  activity 
in  the  chemical  sense,  to  activate  it  in  the  mass  sense.  A  gross 
suggestion  of  such  a  process  is  contained  in  the  proposition  that 
if  a  metal  were  chemically  active  in  itself,  the  finer  the  subdi- 
vision of  the  metal  in  the  system,  the  greater  the  surface  exposed 
for  contact.  Thus  copper  and  platinum  are  slightly  catalytic  in 
sheet  form,  enormously  active  in  colloidal  state.  Whatever  the 
process  may  be,  we  may  be  sure  that  the  catalytic  property  of 
colloidal  suspensions  lies  in  the  chemical  activity  of  the  substance 
in  that  state.  The  physical  properties  of  colloids,  especially  of 
the  stable  organic  colloids,  are  indeed  difficult  of  definition  and 
characterization,  and  almost  impossible  of  control ;  but  that  is  no 
reason  why  the  ' '  colloidal  properties ' '  should  be  blindly  invoked 
as  an  explanation  of  whatever  may  appear  obscure.  The  funda- 
mental fact  in  the  phenomenon  of  fermentation  is  a  chemical  act ; 
and  howsoever  the  physical  conditions  of  the  reacting  substances 
and  the  system  may  modify  that  reaction  in  one  direction  or  an- 
other, they  cannot  supplant  the  chemical  reaction  as  the  funda- 
mental fact  of  the  phenomenon. 


VOL.  1]  Taylor.— On  Fermentation.  295 


LITERATURE. 

Bach  and  Chordat.    Biochemische.  Cenbl.,  1,  417,  457. 

Bodenstein.     Zeitschr.  f.  physik.  Chem.,  46,  725-49,  41,  61. 

Bredig.     Die  Anorganische  Fermente.,  1901. 

Erode.     Zeitschr.  f.  physik.  Chem.,  37,  257-49,  208. 

Engler  and  Weisberg.    Krit.  Studien  u.  d.  Vorg.  d.  Autoxydation,  1904. 

Euler.     Zeitschr.  f.  physik.  Chem.,  36,  641-40,  498-47,  353. 

Berichte,  33,  3202. 

Compare  also  Wegscheider.     Zeitschr.  f.  physik.  Chem.,  39,  257-41,  162. 
Kastle  and  Loevenhart.    Am.  Chem.  Jour.,  29,  397,  563. 
Loew.    Arch.  f.  d.  ges.  Physiol.,  27,  203. 

Science,  1899,  955. 

Luther.     Zeitschr.  f.  physik.  Chem.,  43,  203-46,  777. 
Pollok.     Zeitr.  z.  chem.  Phys.  u.  Path.,  6,  95. 
Schilow.     Zeitschr.  f.  physik.  Chem.,  42,  641. 
Trautz.     Zeitschr.  f.  physik.  Chem.,  47,  513. 
Wegscheider.1    Zeitschr.  f.  physik.  Chem.,  30,  593.    Also  Ibidem.,  34,  290-35, 

565. 

Wegscheider.2    Zeitschr.  f.  physik.  Chem.,  34,  290,  35,  565. 
Kuester.     Zeitschr.  f.  physik.  Chem.,  18,  161. 


296  University  of  California  Publications.       [PATHOLOGY 


THE  SPECIFICITY  OF  FERMENT  ACTION. 

In  the  question  of  the  specificity  of  ferment  action  we  are 
confronted  with  a  problem  of  great  importance.  Like  many 
problems,  it  has  become  appreciable  but  gradually,  with  the  de- 
velopment of  the  collateral  aspects  of  the  general  study  of  fer- 
ments. In  the  routine  text-books  on  physiology  one  may  still 
meet  with  the  statement  that  the  specific  limitations  of  the  power 
of  ferments,  the  ability  to  ferment  but  one  or  at  most  nearly 
allied  bodies,  consitutes  a  distinction  between  ferments  and  in- 
organic catalysors.  Nothing  could  be  farther  from  the  truth. 
There  are  many  instances  of  quite  specific  action  among  inor- 
ganic catalysors.  For  example,  iron  salts  act  as  good  catalysors 
for  the  oxidation  of  potassium  iodide  by  a  persulphate ;  but  they 
will  not  accelerate  the  reaction  of  the  same  persulphate  upon  sul- 
phurous acid.  Wolframic  acid  is  an  active  catalysor  for  the  oxi- 
dation of  hydriodic  acid  by  hydrogen  peroxide,  but  it  will  not 
accelerate  the  same  oxidation  by  a  persulphate.  And  in  a  similar 
manner  potassium  bichromate  will  accelerate  the  oxidation  of 
hydriodic  acid  by  bromic  acid,  but  not  the  oxidation  by  iodic 
acid.  Platinum  black  is  a  good  accelerator  for  the  hydrolysis  of 
esters  of  the  simple  alcohols,  but  it  has  no  appreciable  effect  upon 
the  hydrolysis  of  esters  of  glycerine.  On  the  other  hand,  trypsin 
will  digest  many  different  proteins,  and  even  many  synthetic 
poly-  and  dipeptides.  Laccase  will  accelerate  the  oxidation  of 
many  aromatic  bodies,  and  the  reduction  ferments  will  accelerate 
many  different  reactions  of  reduction.  A  simple  contemplation 
of  the  chemical  relations  concerned  leads  to  the  view  that  since 
these  accelerations  are  to  be  regarded  as  founded  upon  interme- 
diary reactions,  whether  a  ferment  acts  or  not  will  depend  solely 
upon  the  particular  reaction  involved.  The  dissociated  hydrogen 
ions  are  indeed  quite  general  catalysors  for  reactions,  but  they 
do  not  share  this  generality  of  action  with  many  inorganic  ca- 
talysors. An  interesting  exception  to  the  general  rule  that  hy- 
drogen ions  act  as  general  catalysors  is  found  in  the  observation 


VoL-  !]  Taylor. — On  Fermentation.  297 

that  acids  are  not  able  to  convert  adenine  and  guinine  into  xan- 
thin  and  hypoxanthin,  though  these  are  reactions  of  hydrolysis. 
All  catalysors  may  be  said  to  be  more  or  less  specific ;  the  inor- 
ganic catalysors  are  less  specific  (i.e.,  have  a  wider  range  of  avail- 
ability in  the  inauguration  of  intermediary  reactions)  than  the 
organic  ferments.  The  specificity  itself  must  theoretically  be 
vested  in  the  chemical  relations  of  the  intermediary  reactions. 

In  a  discussion  of  the  specificity  of  ferment  action  we  must 
distinguish  between  quantitative  and  qualitative  specificity.  By 
quantitative  specificity  we  mean  whether  a  ferment  does  or  does 
not  accelerate  a  certain  reaction.  By  qualitative  specificity  we 
mean  that  a  ferment  not  only  accelerates  a  reaction,  but  so  modi- 
fies it  as  to  determine  the  chemical  nature  of  the  products. 

In  the  beginning  it  must  be  pointed  out  that  the  fermenta- 
bility  of  a  certain  body  may  be  only  a  relative  term,  with  a  time 
limitation.  When  we  say  that  a  certain  ferment  will  not  act 
upon  a  certain  substance,  we  usually  mean  that  a  test  of  several 
hours  or  days  is  made  and  then  the  results  determined  with  a 
certain  analytical  precision.  The  accuracy  of  the  observation 
depends  upon  the  purity  of  the  reacting  bodies,  the  stability  of 
the  ferment,  the  length  of  time  permitted,  and  the  delicacy  of 
the  analytic  procedures  used  to  determine  the  Recurrence  of  the 
reaction.  It  is  apparent  that  a  ferment  could  act  in  a  positive 
manner,  but  that  the  acceleration  might  not  be  measurable  under 
the  chosen  or  necessary  conditions  of  the  experiment.  In  a  strict 
sense  one  ought  to  demonstrate  that  the  velocity  of  the  reaction 
with  the  ferment  of  supposed  inactivity  possesses  the  same  veloc- 
ity as  the  simple  system  without  ferment.  On  the  other  hand,  a 
positive  result  might  be  spurious.  For  example,  pentoses  are  not 
fermentable  with  zymase ;  but  an  appreciable  quantity  of  alcohol 
and  carbon  dioxide  could  appear  in  such  an  experiment,  derived 
from  the  glycogen  contained  in  the  extract  of  the  yeast.  In  a 
review  of  the  reported  work  one  is  impressed  with  these  facts: 
As  a  rule  the  time  has  been  too  short;  the  analytical  methods  for 
the  determination  of  the  occurrence  of  a  reaction  have  been  often 
coarse ;  and  the  reacting  bodies  and  ferments  have  rarely  been 
pure  enough  to  insure  an  unequivocal  interpretation  of  positive 
or  negative  results.  If  the  successful  reversion  of  ferment  action 


298  University  of  California  Publications.       [PATHOLOGY 

had  been  done  in  the  routine  manner  of  testing  for  ferment  ac- 
tion, not  a  single  one  of  the  now  demonstrated  reversions  would 
have  been  discovered. 

Many  ferments  have  but  a  limited  range  of  activity ;  they  are 
able  to  ferment  but  certain  few  substances.  When  we  say  that 
a  ferment  is  able  to  act  upon  a  certain  body,  we  mean  to  a  meas- 
urable degree.  For  example,  trypsin  is  able  to  ferment  prota- 
mine ;  pepsin  is  not  able  to  ferment  it.  By  this  I  mean  that  in  a 
test  of  several  weeks  no  demonstrable  quantity  of  arginine  may 
be  recovered  from  the  system.  For  many  other  ferments,  how-- 
ever,  the  situation  is  different,  in  that  a  very  slow  fermentation 
occurs.  Thus  pepsin  ferments  reticulin  with  difficulty,  trypsin 
with  still  greater  difficulty,  so  that  the  current  statement  is  that 
reticulin  may  not  be  fermented  with  trypsin.  In  all  probability 
the  true  statement  would  be  that  outside  of  the  fermentations  of 
the  carbohydrates,  which  have  been  best  studied,  all  statements 
of  non-activity  applied  to  ferments  usually  mean  that  under  the 
conditions  of  the  experiment,  in  the  short  life  of  the  ferment,  no 
appreciable  reaction  occurred;  and  it  is  not  equivalent  to  the 
physico-chemical  statement  that  such  and  such  a  ferment  is  not 
a  catalysor  for  such  and  such  a  reaction. 

In  the  case  of  the  sugars,  however,  the  experimental  data  is 
much  greater  in  amount,  and  of  good  quality.  Especially  have 
Emil  Fischer1  and  his  students  worked  with  great  detail  and  deep 
insight  into  these  problems.  He  has  collected  his  experiences 
into  a  general  induction,  which  rests  the  fermentability  of  sugars 
upon  their  own  sterioisomeric  configuration  and  upon  an  appro- 
priate assumed  sterioisomeric  configuration  upon  the  part  of  the 
molecule  of  ferment.  Fischer  studied  early  the  commonly  known 
facts  that  certain  yeasts  will  ferment  only  certain  sugars,  and 
studied  these  relations  in  a  systematic  manner.  He  realized, 
however,  that  no  generalizations  could  be  based  upon  such  living 
experiments  alone,  and  he  repeated  the  experiments  with  the 
powdered  or  expressed  ferments.  He  further  realized  that  to 
make  the  experiments  convincing,  the  configuration  of  the  sugars 
under  study  must  be  undoubted,  and  to  fill  this  requirement  he 
employed  for  his  crucial  experiment  synthesized  sugars. 


VOL- 1]  Taylor. — On  Fermentation.  299 

The  fermentability  of  a  sugar  depends,  according  to  this 
hypothesis,  upon  the  sterioisomeric  configuration  of  its  own  mole- 
cule and  of  the  molecule  of  the  ferment.  This  statement  is  in 
reality  not  revolutionary.  Many  facts  in  the  chemistry  of  the 
sugars  indicate  that  the  resistance  to  reactions  and  the  reaction 
ability  is  allied  not  solely  to  the  structural,  but  also  the  sterio- 
isomeric configuration.  The  different  hexoses  present  widely 
varying  relations  to  the  different  compounds  of  hydrazine ;  the 
different  osazones  and  hydrozones  vary  widely  in  their  solubility, 
velocity  of  formation,  stability,  etc.  The  resistance  of  different 
sugars  towards  simple  reagents  like  acids  display  also  variations. 
Thus  maltose  is  most  easily  hydrolyzed  by  acid,  cane  sugar  next, 
and  lactose  most  difficultly  of  all.  Two  molecules  of  d-glucose 
unite  to  form  a  disaccharide,  maltose;  but  two  molecules  of  d- 
laevulose  or  of  d-galactose  do  not  unite  to  form  disaccharides, 
though  each  of  them  will  unite  with  d-glucose  to  form  disaccha- 
rides, but  do  not  unite  with  each  other.  From  the  point  of  view 
of  fermentations  as  accelerations  through  intermediary  reactions, 
the  Fischer  hypothesis  is  very  feasible,  since  the  configuration 
might  naturally  be  supposed  to  be  of  marked  or  even  dominating 
influence  in  such  intermediary  reactions.  The  specificity  of  the 
ferment  lies  in  the  coadaptation  of  the  configuration  of  a  partic- 
ular ferment  for  certain  sugars,  just  as  hydrogen  disulphide  is  a 
specific  precipitant  for  certain  groups  of  metals. 

For  the  members  of  the  benzol  series  a  large  number  of  in- 
stances are  known  in  which  reaction  affinity  is  dependent  upon 
or  associated  with  a  certain  configuration.  The  location  of  radi- 
cals in  a  benzol  body  determines  often  the  resistance  to  chemical 
reaction  displayed  by  that  body,  in  that  the  substitution  of  hy- 
drogen is  not  effected  with  the  same  readiness  when  the  radicals 
occupy  different  positions.  Thus  substitution  by  sulphur  radi- 
cals is  easy  in  meta-xylol,  less  ready  in  ortho-xylol,  and  difficult 
in  para-xylol.  In  the  case  of  substitution  by  nitric  acid,  on  the 
contrary,  as,  for  instance,  in  the  action  of  nitric  acid  upon  the 
isomeric  nitrotoluols,  the  reaction  is  most  easy  in  the  ortho-  and 
most  difficult  in  the  meta-nitrotoluol.  The  oxidation  of  a  lateral 
chain  to  a  carboxyl  group  is  likewise  related  to  the  configuration ; 
ortho  derivatives  resist  the  action  of  chromic  acid  entirely,  while 


300  University  of  California  Publications.       [PATHOLOGY 

para  derivatives  are  quite  susceptible;  thus  ortho-brom-benzyl- 
bromide  is  entirely  refractory  to  chromic  acid,  while  the  para- 
brom-benzyl-bromide  is  easily  oxidized.  And  for  the  same  reason 
benzyl-chloride  is  more  easily  oxidized  to  benzoic  acid  than  is 
toluol.  Similar  relations  exist  for  the  splitting-  off  of  the  car- 
boxyl  group:  in  ortho-  and  para-oxybenzoic  acid  this  may  be  ac- 
complished by  hydrochloric  acid,  which  will,  however,  fail  with 
the  meta-oxybenzoic  acid.  In  the  case  of  the  chlorhydrates  of 
nitro-anilines.  the  dissociation  varies;  at  ordinary  temperature 
the  ortho  derivative  is  dissociated  to  10  per  cent.,  the  para  IP  5 
per  cent.,  and  the  meta  derivative  to  but  less  than  1  per  cent. 
In  an  analag-ous  manner,  the  catalytic  action  of  metals  in  syn- 
thetic reactions  with  aromatic  bodies  illustrates  a  certain  speci- 
ficity, thus  in  the  sulphuring-  of  anthrachinons  in  the  presence  of 
the  salts  of  mercury,  sulpho  acids  of  the  a  series  are  formed 
in  larp-e  part,  which  is  not  true  in  the  presence  of  other  heavy 
metals.  Another  illustration  is  the  action  of  boric  acid  in  the 
synthesis  of  poly-oxy-anthrachinons.  Not  only  is  there  a  relation 
of  specificity  between  the  configuration  of  the  reacting-  aromatic 
body  and  the  metal,  there  is  also  a  specificity  in  the  resulting- 
product,  and  in  a  general  sense  under  these  circumstances  these 
metals  might  be  spoken  of  as  catalysors  that  not  only  accelerate 
the  velocity  of  the  reaction  but  also  modify  the  products.  Illus- 
trations could  be  adduced  in  numbers  from  the  chemistry  of  the 
benzene  series  in  which  the  interreaction  of  two  ring-  compounds 
is  dependent  upon  an  appropriate  configuration  of  the  two  mole- 
cules. 

A  very  striking-  illustration  of  the  relations  of  reaction  accel- 
eration to  configuration  is  to  be  noted  in  the  recent  studies  in 
photochemistry.  The  results  of  the  studies  of  Ciamician  and 
Sachs  and  their  respective  students  seems  to  point  to  the  fact 
that  the  sensibility  to  light  upon  the  part  of  aromatic  bodies  is 
noted  only  in  such  bodies  as  possess  a  nitro  group  in  the  ortho 
position  to  a  CH^group.  Now  many  of  these  bodies  are  ferment- 
able, and  the  fermentative  accelerations  bear  similar  relations  to 
the  configuration.  Thus  laccase  will  accelerate  the  oxidation  of 
hydrochinon  (para-dioxybenzol),  but  will  not  ferment  the  ortho- 
(pyro-catechin)  or  the  meta-dioxybenzol  (resorcin).  Tyrosinase, 


VOL.  l]  Taylor. — On  Fermentation.  301 

furthermore,  will  ferment  metatoluidine,  but  not  the  ortho-  or 
para-toluidine,  while  it  will  ferment  all  three  of  the  zylenols. 

If  now  invertase  be  supposed  to  possess  a  sterioisomeric  con- 
figuration simply  because  it  bears  experimentally  specific  rela- 
tions to  the  fermentation  of  particular  sterioisomeric  molecules 
of  sugar,  laccase  and  tryosinase  might  be  supposed  to  bear  certain 
configurations  corresponding  to  the  ring  structures  of  the  aro- 
matic bodies  they  ferment.  Yet  in  the  case  of  these  t\vo  last 
ferments  this  cannot  be  conceded,  because  identical  specific  rela- 
tions apply  to  chromic,  nitric,  and  sulphuric  acids,  which  can 
possess  no  such  thing.  This  same  general  consideration  will 
apply  to  large  numbers  of  ferments,  like  urease,  arginase,  lipase, 
animal  oxidase,  the  acetic  acid  fermentation  of  alcohol  and  alde- 
hyde, nitrification  and  denitrification,  the  bacterial  reductions 
of  metallic  oxides  or  salts.  In  all  of  these  such  a  relation  be- 
tween configuration  of  substrate  and  ferment  cannot  be  claimed. 
Fischer  has  himself  never  claimed  for  his  theory  that  it  would 
fit  all  classes  of  fermentations,  though  this  has  been  almost  un- 
consciously assumed  by  nearly  all  of  the  writers  upon  the  subject, 
particularly  by  the  biologists. 

Other  suggestive  illustrations  of  the  relations  between  config- 
uration and  reaction  ability  are  to  be  seen  in  the  esterification 
of  different  benzoic  acids.  As  Victor  Meyer  has  shown,  the  re- 
placement of  the  hydrogen  atoms  in  the  ring  results  in  a  reduc- 
tion of  the  tendency  to  the  reaction,  due  to  the  absence  of  the 
hydrogen,  which  accelerates  reaction.  The  measurement  of  the 
reaction  is  accomplished  by  introducing  the  benzoic  acid  into  an 
excess  of  methyl  alcohol  saturated  with  hydrochloric  acid.  But 
the  relations  of  the  different  hydrogens  are  not  identical.  The 
carboxyl  group  being  placed  in  the  position  of  1  in  the  ring,  if 
the  hydrogens  are  replaced  from  2  and  4,  or  from  3,  4,  and  5, 
over  95  per  cent,  of  ester  will  be  formed ;  while  if  the  two  hy- 
drogens adjacent  to  the  carboxyl  group  are  replaced,  at  2  and  4, 
or  at  2,  4,  and  6,  almost  no  ester,  less  than  5  per  cent.,  will  be 
formed.  Of  influence  further  is  the  mass  of  the  radicle  that  re- 
places the  hydrogen  in  the  ortho  position  to  the  carboxyl  group ; 
thus  bromine  and  iodine  with  their  heavy  molecular  weights  de- 
press the  esterification,  while  methyl  has  but  slight  inhibitory 


302  University  of  California  Publications.       [PATHOLOGY 

effect.  The  influence  of  the  substitution  of  the  hydrogen  in  the 
ortho  position  has  been  also  well  shown  in  the  studies  of  Gold- 
schmidt,  who  showed  also  that  when,  as  in  phenyl  acetic  acid, 
hydrogens  were  attached  to  both  the  carbons  that  carried  the  car- 
boxyl  groups,  the  reaction  velocity  was  high,  while  in  benzoic 
acid,  which  has  no  such  hydrogens,  the  reaction  velocity  was  low, 
only  about  1  per  cent,  of  that  of  phenyl-acetic  acid.  These  va- 
rious relations  are  probably  best  interpreted  to  mean  that  sub- 
stitution of  hydrogen  by  radicles  results  in  increased  resistance 
to  reaction,  and  that  this  resistance  is  very  different  for  different 
positions  in  the  ring.  That  analogous  relations  hold  for  simple 
compounds  is  to  be  noted  in  the  fact  that  fumaric  acid  does  not 
tend  to  the  formation  of  the  anhydride,  while  the  isomeric  maleic 
acid  does  tend  to  the  formation  of  the  anhydride ;  this  van 't  Hoff 
ascribes  to  the  fact  that  in  maleic  acid  the  carboxyl  groups  are 
adjacent,  while  in  the  fumaric  acid  they  are  separated.  This  re- 
lation of  the  tendency  to  anhydride  formation  to  the  relative 
situations  of  the  carboxyl  groups  seems  to  hold  in  many  com- 
pounds. Another  illustration  of  the  relation  between  configura- 
tion and  reaction  ability  has  been  furnished  by  Fischer  himself, 
who  has  shown  that  in  compounds  that  are  asymmetric  by  reason 
of  the  attachment  of  a  glucose  fraction,  radicles  such  as  HCN 
may  be  attached  in  an  asymmetric  manner  to  the  carbonyl  group. 
The  theory  of  Fischer  rests  upon  the  general  proposition  that 
for  the  production  of  an  optically  active  substance  the  interven- 
tion of  an  asymmetric  optically  active  substance  is  necessary. 
The  accepted  hypothesis  bearing  upon  the  derivation  of  the  asym- 
metric sugars  and  glucoses  in  nature  rests  their  synthesis  upon 
a  photochemic  process.  The  solar  light  is  known  to  contain  linear 
polarized  light  at  the  surface  of  our  sphere.  When  this  light 
impinges  upon  the  surface  of  the  seas,  a  certain  production  of 
circumpolarization  is  held  to  occur.  The  relations  of  the  earth's 
magnetism  are  held  accountable  for  quantitative  dispersion  of 
this  circumpolarized  light.  The  action  of  this  light  is  held  to 
account  for  the  synthesis  of  asymmetric  and  therefore  optically 
active  carbohydrates.1  Whether  the  synthesis  of  asymmetric  su- 
gars occurs  directly  in  the  plants  under  the  influence  of  circum- 
polarized light,  or  through  the  agency  of  asymmetric  substances 


VOL.  1]  Taylor.— On  Fermentation.  303 

in  the  chlorofyl  granules,  as  Fischer  believes,  is  immaterial  to 
the  general   theory,   since  the   asymmetric   chlorofyl  substance 
would  need  to  be  derived  in  the  same  manner  through  the  agency 
of  cireumpolarized  light.     Of  course  the  converse  of  the  propo- 
sition that  only  asymmetric  bodies  can  produce  an  asymmetric 
substance — that  only  an  asymmetric  body  can  build  down  an- 
other asymmetric  body — is  not  stated.    But  among  the  reaction 
possibilities  of  an  asymmetric  substance  we  might  most  reason- 
ably suppose  that  some  reaction  tendencies  as  well  as  resistance 
to  reaction  might  be  vested  in  the  sterioisomeric  configuration, 
and  would  also  correspond  to  the  sterioisomeric  configuration  of 
the  second  reacting  molecule.     This  relation  of  the  two  reacting 
molecules  Fischer  compared  to  the  relations  between  a  lock  and 
key :  and  as  a  purely  symbolic  illustration  he  compared  the  lat- 
eral recessions  of  the  lock  and  the  lateral  projections  of  the  key 
to  the  lateral  arrangements  of  the  elements  upon  the  carbon  chain 
of  a  sugar  molecule.    For  Fischer  this  arrangement  meant  simply 
a  correlation  favorable  to  interreaction  in  the  purely  chemical 
sense.     It  has  been  unfortunate  for  the  future  development  of 
our  knowledge  of  this  and  allied  subjects  that  the  chemical  term- 
inology was  appropriated  by  Ehrlich  and  applied  to  phenomena 
of  a  different  order  and  magnitude,  of  which  the  physical  and 
chemical  properties  are  almost  entirely  unknown.     As  Ostwakl 
has  put  it,  the  lock  and  key  hypothesis  is  being  applied  to  all 
sorts  of  phenomena,  because  everybody  knows  what  lock  and  key 
are.     Indeed.  Ehrlich  has  admitted  that  his  use  of  the  term  lat- 
eral chain   is  philological  and  not  chemical.     The  Ehrlich  hy- 
pothesis is  not  a  physical  or  chemical  hypothesis  at  all,  but  sim- 
ply a  verbal  scheme  of  explanation  along  the  line  of  least  resist- 
ance, the  true  verbality  of  which  has  become  fully  apparent  only 
since  some  of  the  phenomena  concerned  have  been  subjected  to 
objective  physico-chemical  investigation,  and  since  Ehrlich  has 
expanded  his  hypothesis  to  attempt  to  explain  with  it  all  the 
nutritional  processes  of  the  body. 

In  another  direction  the  wording  of  the  Fischer  hypothesis 
and  in  particular  its  interpretation  by  the  biological  world  has 
been  unforunate.  Although  Fischer  does  not  state  so  specifi- 
cally, the  general  wording  of  his  writings  suggests  that  it  is  the 


304  University  of  California  Publications.       [PATHOLOGY 

presence  of  the  ferment  that  inaugurates  the  reaction  of  fermen- 
tation. One  would  assume  from  the  descriptions  given  that  when 
the  isomeric  methyl-glucosides  are  tested  with  the  different  fer- 
ments, it  is  the  ferment  that  gives  the  occasion  for  the  installa- 
tion of  the  reaction.  Stress  is  constantly  laid  on  the  sterioiso- 
meric  configuration  in  the  positive  sense,  in  the  direction  of  the 
faculty  of  reaction.  Now  these  reactions  are  theoretically  and 
experimentally  known  to  be  not  new,  but  accelerated  reactions ; 
the  ferment  does  not  act  as  a  key  to  open  a  molecule  to  a  new 
reaction.  The  influence  of  the  sterioisomeric  configuration  lies, 
in  the  opposite  direction.  Certain  configurations  endow  the  mole- 
cule with  a  great  marked  resistance  to  the  reaction ;  the  ferment 
modifies  this  internal  resistance.  The  facts  are  of  course  the  same 
in  both  instances,  but  the  point  of  view  is  important.  The  reac- 
tion between  a  toxine  and  an  antitoxine  is  of  course  a  reaction 
de  novo,  and  this  constitutes  a  fundamental  difference  between 
it  and  a  fermentation.  Despite  this,  however,  the  Fischer  hy- 
pothesis has  been  transferred,  in  the  verbal  sense  at  least,  to  this 
phenomenon ;  and  lately  the  Ehrlich  interpretations  of  the  facts 
in  the  domain  of  toxines  and  antitoxines  have  been  urged  in  the 
attempt  to  explain  the  facts  in  fermentations.  This  incongruity 
has  arisen  largely  from  the  neglect  of  the  principle  that  the  fer- 
ment or  catalysor  deals  not  with  the  impelling  force  of  a  reaction, 
but  solely  with  the  internal  passive  resistance  to  a  reaction.  It 
has  been  this  same  misunderstanding  that  has  led  biologists  to 
deny  the  fermentative  acceleration  of  the  reversed  reaction.  No- 
\vhere  in  the  writings  of  Fischer  is  it  stated  that  fermentations 
are  reactions  de  novo  and  not  accelerated  reactions ;  but  the  cur- 
rent biological  interpretation  of  his  hypothesis  is  as  stated. 

There  are  sixteen  possible  sterioisomerides  of  hexose.  Of 
these  twelve  have  been  isolated  from  natural  sources  or  synthe- 
sized. Of  these  but  four  are  susceptible  of  alcoholic  fermenta- 
tion by  zymase :  d-glucose,  d-mannose,  d-laevulose,  and  d-galac- 
tose.  For  purposes  of  illustration,  the  sterioisomeric  configura- 
tions of  these  will  be  given,  together  with  that  of  d-talose,  which 
is  not  fermentable. 


VOL-  !]  Taylor. — On  Fermentation.  305 

d-glucose  d-mannose  d-fruetose  d-galaetose  d-talose 

COH  COH  -CH2.OH  COH  COH 

I  I  I  I  I 

H.    C.OH  HO.C.H  CO  H.C.OH  HO.C.H 

I  I  I  I  | 

HO.C.H  HO.C.H  HO.C.H  HO.C.H  HO.C.H 

I  I  I  I  I 

H.    C.OH  H.C.OH  H.C.OH  HO.C.H  HO.C.H 

I  I  I  I  I 

H.    C.OH  H.C.OH  H.C.OH  H.C.OH  H.C.OH 

CH2.OH  CH,.OH  CH..OH  CH,.OH  CH,.OH 


The  d-glucose,  d-laevulose,  and  d-mannose  are  identical  in 
this,  that  the  relations  of  the  three  asymmetric  atoms  of  carbon 
that  are  common  to  them  all  are  alike.  The  other  asymmetric 
atom  in  the  d-glucose  and  d-mannose  seems  to  be  of  no  determin- 
able  influence.  In  the  molecule  of  the  d-galactose  there  is  the 
difference  from  the  other  three,  that  in  the  center  one  of  the  three 
asymmetric  atoms  of  carbon  common  to  them  all,  the  center  car- 
bon has  the  relations  of  hydrogen  and  hydroxyl  reversed  laterally. 
The  upper  asymmetric  carbon  is  identical  in  its  relations  with 
the  corresponding  atom  of  the  d-glucose,  but  has  the  reverse  re- 
lation of  lateral  attachments  possessed  by  the  d-mannose.  Never- 
theless these  differences  are  not  determinating,  since  d-galactose 
is  fermentable  by  the  same  yeasts,  though  some  yeasts  cannot 
ferment  it  at  all,  and  all  do  so  with  slower  velocity.  It  is  there- 
fore not  the  situations  of  individual  hydroxyls  that  determine 
the  fennentability,  but  the  total  combination.  Thus  d-talose, 
which  is  non-fermentable,  resembles  d-mannose  in  the  relations 
of  the  upper  asymmetric  carbon,  d-galactose  in  the  relations  of 
the  two  middle  asymmetric  carbons,  and  all  four  in  the  relations 
of  the  lower  asymmetric  carbon.  A  direct  rule  is  obviously  not 
contained  in  these  facts,  but  somewhere  in  the  series  of  asym- 
metric carbon  atoms  of  d-talose  is  a  high  passive  resistance  not 
encountered  in  the  others,  while  somewhere  in  the  series  of  asym- 
metric carbons  in  d-galactose  is  a  certain  degree  of  passive  re- 
sistance not  met  with  in  the  other  three.  There  can  be  no  doubt 
that  the  facts  suggest  a  quantitative  rather  than  a  qualitative 
difference.  In  the  original  hypothesis  it  was  postulated  that  the 
sugars  with  carbons  in  multiple  of  three  should  be  fermentable 
by  the  same  yeasts.  In  this  point,  however,  the  hypothesis  failed 


306  University  of  California  Publications.       [PATHOLOGY 

outright.  The  nonoses  do  not  ferment,  while  the  apparent  fer- 
mentation of  the  aldo-glycerose  and  the  keto-glycerose  were 
shown  to  have  been  due  to  condensations  into  hexoses.  (Piloty, 
Wohl,  Emmerling.1)  That  the  pentoses  and  other  sugars  do  not 
undergo  alcoholic  fermentation,  as  usually  stated,  is  not  so  cer- 
tain. That  pentoses  are  very  resistant  to  alcoholic  fermentation 
is  true.  Nevertheless  it  is  known  that  certain  bacteria  do  pro- 
duce in  arabinose  fermentations  that  are  accompanied  by  the 
formation  of  alcohol.  These  fermentations  seem  always  mixed, 
so  far  as  the  products  are  concerned;  lactic,  acetic,  formic,  and 
butyric  acids  in  particular  are  also  present.  (Frankland  and 
MacGregor,  Salkowski,  Tollens  and  Schoene,  Harden.)  One 
must  assume,  if  one  clings  to  the  assumption  that  the  pentose  is 
resistant  to  alcoholic  fermentation,  that  the  alcohol  under  these 
circumstances  is  derived  secondarily  from  the  real  products  of 
the  fermentation.  More  proper  chemically  would  it  be  to  assume 
that  pentose  is  susceptible  of  alcoholic  fermentation  by  certain 
yeasts,  but  the  side  reactions  complicate  the  products;  thus  the 
lactic  acid  could  be  the  result  of  an  incomplete  reaction,  and  from 
it  the  butyric  acid  could  be  derived,  while  the  acetic  acid  would 
represent  the  oxidation  product  of  alcohol. 

So  far  as  the  alcoholic  fermentation  of  sugars  is  concerned 
we  may  therefore  say  that  of  the  three  propositions — that  all 
sugars  with  carbon  atoms  in  multiples  of  three,  and  only  these 
are  fermentable,  that  of  the  hexoses  only  the  d-members  of  the 
sterioisomeric  series  are  fermentable,  and  of  these  only  those 
possessing  a  certain  configuration  are  fermentable — the  first  is 
not  correct,  the  second  is  correct  so  far  as  known,  and  the  third 
is  correct  in  the  quantitative  sense,  though  improved  in  the  quali- 
tative sense. 

Cremer  has  advanced  a  suggestion  that  locates  the  fermenta- 
bility  of  the  hexoses  in  their  being  convertible  into  d-glucose.  He 
suggests  that  only  d-glucose  is  fermentable,  and  those  sugars  only 
subject  to  fermentation  that  are  convertible  into  d-glucose.  That 
these  hexoses  may  be  easily  converted  into  each  other  through  the 
action  of  alkali  has  been  shown  by  Lobry  du  Bruyn  and  van  Eren- 
stein.  The  formation  of  d-laevulose  from  d-glucose  is  according 
to  recent  investigations  of  Ost  not  confined  to  the  experimental 


VOL.  1]  Taylor.— On  Fermentation.  307 

conditions  described  by  these  authors,  but  occurs  in  every  hy- 
drolysis of  starch,  arising  not  from  the  starch  directly  but  by 
polymerization  of  the  d-glucose.  The  theory  of  Cremer  has  no 
direct  or  experimental  proof  in  its  favor,  and  is  opposed  by  many 
demonstrated  facts. 

In  the  attempt  to  secure  clearer  relations,  Fischer2  tested  the 
fermentation  of  natural  and  synthetic  glucosides.  When  aldo- 
hexoses  are  heated  in  alcoholic  hydrochloric  acid,  glucosides  are 
formed.  Of  both  the  d-  and  1-sugars  two  isomers  are  formed, 
termed  the  a-  and  b  series.  Thus  for  methyl  alcohol  we  have  the 
two  isomers  of  d-glucose : 


H.C.OCH.3 


H.C.OH 

I 
HO.C.H 


Fischer  found  that  the  glucosides  of  the  1-series  of  glucose, 
mamiose,  and  galactose  were  not  fermentable  at  all.  The  gluco- 
sides of  the  d-series  of  glucose,  mannose,  and  galactose  were  fer- 
mentable, but  to  a  noteworthy  degree  specifically.  The  a-gluco- 
sides  of  these  d-hexoses  were  fermentable  with  yeasts,  the  &-glu- 
cosides  were  fermentable  with  emulsine.  The  natural  glucosides 
are  &-glucosides.  so  that  the  concordance  between  the  natural  and 
the  synthetic  glucosides  is  complete.  Amygdaline,  however,  is 
split  by  the  alcoholic  yeast  to  the  extent  that  one  molecule  of 
d-glucose  is  split  off;  the  remaining  mandelnitril-glucoside,  how- 
ever, is  fermentable  only  by  emulsine.  Whether  the  emulsine  is 
able  to  accomplish  the  complete  fermentation  of  the  amygdaline, 
or  whether  a  maltase  is  always  present,  is  not  determined.  It  is 
not  the  zymase  in  the  yeast  that  accelerates  these  reactions ;  it  is 
the  maltase  or  allied  ferment.  The  data  bearing  on  these  syn- 
thetic glucosides  is  very  positive,  and  suggests  strongly  the  va- 
lidity of  the  Fischer  hypothesis.  The  time  allowed  the  tests  has, 
however,  been  usually  short,  often  only  twenty  hours,  so  that  a 


308  University  of  California  Publications.       [PATHOLOGY 

slight  acceleration  of  the  cleavage  of  the  6-glucosides  by  the  mal- 
tase  and  the  a-glueosides  by  the  emulsine  was  not  positively  ex- 
cluded. 

Similar  considerations  hold  for  the  disaccharides.  Invertase 
ferments  cane  sugar,  and  is  depressed  only  by  the  d-laevulose, 
not  by  the  d-glucose.  Maltose  ferments  only  maltose,  and  is  de- 
pressed of  course  by  the  d-glucose.  Neither  of  these  ferments 
can  split  a  typical  &-glucoside  or  milk  sugar.  Lactase  ferments 
milk  sugar,  and  also  &-glucosides,  but  no  er-glucosides ;  it  is  de- 
pressed only  by  the  d-galactose.  Milk  sugar  for  its  part  is  fer- 
mentable by  emulsine,  which  is  able  to  ferment  neither  cane 
sugar  nor  maltose.  This  seems  strange,  since  maltose  and  lactose 
are  structurally  so  closely  related,  and  have  apparently  closely 
corresponding  sterioisomeric  configurations.  Pottevin  has  re- 
cently repeated  the  experiments,  and  could  not  find  that  milk 
sugar  was  fermented  by  emulsine  or  that  lactase  was  able  to 
ferment  Z>-glueosides.  This  would  accord  much  better  with  the 
Fischer  theory  than  the  earlier  results  of  Fischer  himself.  As 
a  matter  of  fact,  these  and  all  the  other  experiments  ought  to  be 
repeated  with  isolated  purified  ferments,  under  careful  condi- 
^tions  of  experimentation. 

Confirmatory  evidence  has  been  very  recently  obtained  by 
Fischer3  through  the  study  of  the  tryptic  digestion  of  synthetic 
peptides.  Of  the  many  synthetic  peptides  that  have  been  pre- 
pared and  tested  by  Fischer  and  his  pupils,  the  following  racemic 
substances  are  digestible  with  trypsin :  Alanyl-glycin,  alanyl- 
alanine,  alanyl-leucine  A,  leucyl-isoserine,  alanyl-glycyl-glycin, 
leucyl-glycyl-glycin,  glycyl-leucyl-alanine,  and  alanyl-leucyl-ala- 
nine.  When  now  these  racemic  peptides  are  digested  with  tryp- 
sine,  the  cleavage  is  an  asymmetric  one,  and  the  amido  acid  that 
is  split  off  is  the  same  active  amido  acid  that  is  to  be  found  among 
the  products  of  the  digestion  of  natural  protein  by  trypsine. 
There  are  two  racemic  alanyl-leucine  peptides,  and  they  include 
the  four  possible  combinations  of  the  two  components.  Thus 
alanyl-leucine  A  is  d-alanyl-1-leucine  -\-  1-alanyl-d-leucine ;  while 
B  is  d-alanyl-d-leucine  -f-  1-alanyl-l-leucine.  Only  A  is  digest- 
ible, and  of  the  compound  only  the  d-alanyl-1-leucine  is  split  off, 
and  the  two  components  separated.  These  facts  have  all  the  more 


VOL.  1]  Taylor.— On  Fermentation.  309 

weight,  because  they  have  been  obtained  from  a  class  of  com- 
pounds totally  different  from  the  sugars. 

According  to  the  theory  of  Fischer,  since  the  velocity  of  re- 
action under  the  influence  of  a  ferment  depends  upon  the  sterio- 
isomeric  configuration  of  the  ferment  molecule,  the  influence  of 
organic  acids  upon  the  same  reactions  should  depend  upon  sim- 
ilar relations.  Fischer*  himself  tested  this  theory.  He  studied 
the  acid  hydrolysis  of  cane  sugar  under  the  influence  of  d-  and 
1-camphoric  acid;  the  result  was  negative  to  his  theory,  for  the 
acceleration  was  identical  in  the  two,  i.e.,  the  acceleration  de- 
pended upon  the  electrolytic  dissociation,  and  not  upon  the  opti- 
cal isomerism. 

Another  interesting  point  from  which  to  view  the  theory  lies 
in  the  fermentation  of  racemic  acids.  Pfeffer,  Purdie,  and  more 
recently  Mackenzie  and  Harden  have  gone  over  this  ground  quite 
thoroughly.  Their  results  are  in  the  main  in  accord,  in  that  both 
the  d-  and  1-acids  are  fermented,  but  with  different  velocities. 
In  some  few  instances  the  reactions  upon  the  two  enantiomorphic 
bodies  were  quite  equal,  in  other  instances  there  was  a  distinct 
predominance;  in  a  few  instances  the  reaction  with  the  one  was 
marked,  with  the  other  very  slight.  The  greater  velocities  were 
in  the  direction  demanded  by  the  Fischer  theory.  If  these  re- 
sults may  be  applied  to  the  fermentation  of  sugars,  it  indicates 
that  the  specificity  is  only  one  of  degree,  that  it  is  not  a  question 
of  reaction  or  no  reaction,  but  of  slight  reaction  as  against  pro- 
nounced reaction. 

Condelli  has  shown  that  the  temperature  optimum  is  different 
for  the  d-  and  1-acids.  He  suggested  further  that  a  particular 
yeast  were  able  to  act  upon  the  1-acid  only  by  first  converting  it 
into  the  d-acid.  This  is  begging  the  question. 

Dakin  has  studied  the  cleavage  of  optically  inactive  racemic 
synthetic  esters  by  lipase.  The  products  were  found  to  be  rotary 
in  one  direction,  and  the  residual  ester  was  rotary  in  the  oppo- 
site direction.  In  a  word,  the  cleavage  was  an  asymmetrical  pro- 
cess. In  the  various  esters  studied,  those  optical  isomerides  most 
rapidly  attacked  possessed  similar  configurations,  thus  conform- 
ing to  the  Fischer  theory.  But  it  was  simply  a  difference  of 
velocity,  not  a  qualitative  differentiation.  The  corresponding 


University  of  California  Publications.       [PATHOLOGY 

fact  to  this  is  to  be  found  in  the  observation  of  Markwald  and 
Mackenzie  that  two  optically  opposite  active  acids  do  not  form 
ester  with  an  optically  active  alcohol  with  the  sarje  velocity.  The 
unequal  velocities  are  explained  by  Dakin  as  follows:  When 
optical  isomerides  combine  separately  with  the  same  structurally 
asymmetric  substance,  they  may  do  so  with  unequal  velocities; 
and  conversely,  the  products  formed  by  such  reactions,  since  they 
are  no  longer  optical  opposites,  might  be  expected  to  undergo 
further  changes  at  unequal  velocities.  If  the  enzyme  be  sup- 
posed to  be  dextro-rotary  and  the  components  of  the  racemic 
acid  be  represented  by  +  S  and  —  8,  the  additive  compounds 
formed  by  the  union  of  the  ester  and  the  ferment  would  be 
(-fe-f  S)  and  (-fe  —  S).  These  two  compounds  are  ob- 
viously not  enantiomorphic  (opposite  compounds  would  be  re- 
spectively (— e  — S)  and  (—  e  +  S),  and  might  therefore  be 
expected  to  be  formed  and  undergo  changes  at  different  veloc- 
ities. This  argument  may  be  pursued  further.  Ferments  are 
apparently  sometimes  active,  sometimes  racemic  substances.  If 
a  racemic  ferment  react  with  a  racemic  substrate,  we  would  ex- 
pect the  formation  of  enantiomorphic  complexes  of  ferment- 
substrate,  and  under  these  circumstances  we  should  expect  the 
reaction  relations  to  be  identical.  Under  these  circumstances  we 
should  expect  an  active  acid  to  act  in  a  different  manner  from  a 
racemic  or  an  inactive  acid.  Fischer  tested  this  hypothesis,  by 
the  use  of  active  camphoric  acid  in  the  inversion  of  sugars,  with 
negative  results,  as  previously  stated. 

There  is,  on  the  contrary,  considerable  data  bearing  against 
the  Fischer  hypothesis.  For  the  bacterial  fermentations,  and 
especially  for  the  lactic  acid  fermentations,  results  have  been 
obtained  that  are  not  in  harmony  with  the  theory.  Emmerling,2 
a  pupil  of  Fischer,  in  his  brochure  on  the  fermentation  of  carbo- 
hydrates, repudiates  squarely  the  position  that  a  certain  germ  in 
pure  culture  will  under  all  circumstances  with  a  known  sugar 
as  substrate  produce  the  same  reaction.  Upon  this  aspect  of  the 
question,  however,  little  weight  is  to  be  laid.  The  theory  of 
Fischer  cannot  be  proved  or  disproved  by  cultural  investigations ; 
isolated  purified  ferments  can  alone  furnish  data  of  the  quality 
necessary  in  deciding  a  fundamental  problem.  A  theory  of  fer- 


VOL.  i]  Taylor. — On  Fermentation.  311 

mentation  it  is  not  and  in  the  nature  of  things  cannot  be.  When 
it  is  shown  that  for  a  particular  fermentation  the  intermediary 
reactions  that  constitute  the  acceleration  are  dependent  upon  the 
possession  of  a  certain  sterioisomeric  or  other  configuration  upon 
the  part  of  the  substrate  or  the  substrate  and  ferment,  that  con- 
stitutes a  fact  for  that  fermentation.  The  investigations  of  the 
future  must  determine  how  closely  the  facts  conform  to  the  the- 
ory in  a  particular  group  of  fermentations,  and  to  how  many 
classes  of  fermentations  the  principle  is  applicable.  It  will  quite 
certainly  be  found  that  the  relation  is  a  quantitative  one,  not  a 
qualitative  one.  A  certain  configuration  is  favorable  to  the  reac- 
tion, another  one  much  less  favorable.  Just  as  the  ferments  are 
tested  on  a  particular  sugar,  so  acids  may  be  tested.  Mineral 
acids  would  give  results  writhin  a  few  moments.  Some  weak  or- 
ganic acids  would  not  give  measurable  results  for  days.  But  the 
actions  of  the  different  acids  are  identical  in  quality,  though 
varying  greatly  in  quantity.  Similar  relations  may  theoretically 
be  expected  from  the  ferments. 

The  reason  a  ferment  is  elective  in  the  sense  of  Fischer  must 
be  ascribed  to  the  fact  that  configuration  means  resistance  to  the 
reaction  or  that  it  means  resistance  to  the  catalysor.  van't  Hoff 
has  classified  the  inhibitory  properties  in  substances  that  operate 
against  the  reaction  velocity.  These  find  unquestionably  appli- 
cation to  the  present  problem.  They  are : 

(a)  Nature  of  the  inhibitory  influences  in  changes  in  physical 
state.  (1)  The  necessity  of  molecular  orientation.  (2)  Neces- 
sity of  spacial  transition.  (3)  Capillary  influences. 

(6)  Nature  of  inhibitory  influences  in  chemical  transforma- 
tions. (1)  The  necessity  of  molecular  orientation.  (2)  The  ne- 
cessity of  special  transition.  (3)  Capillary  influences.  (4)  In- 
hibitory influences  of  undefined  nature  that  are  operative  for 
certain  reactions  and  characteristics  of  them,  independent  of  the 
previously  enumerated  factors.  Of  these  two  illustrations  may 
be  given.  Fumaric  acid  under  certain  conditions  remains  stable 
and  does  not  pass  into  maleic  acid,  though  it  is  easy  to  show  that 
the  latter  is  the  more  stable  form,  and  easily  produced  from  the 
former  by  appropriate  contact  action.  Here,  in  short,  an  auto- 
reaction  does  not  occur  with  any  velocity.  Secondly,  we  know 


312  University  of  California  Publications.       [PATHOLOGY 

that  there  are  for  many  substances  and  complexes  conditions, 
especially  known  for  temperature  and  pressure,  under  which  the 
substances  remain  in  a  state  of  apparent  equilibrium,  which 
may,  however,  be  demonstrably  not  the  true  equilibrium.  Pe- 
labon  has  shown  that  the  system  SeH2  =  Se  -f  H2  forms  above 
325°  a  true  state  of  equilibrium;  it  is  immaterial  from  which 
direction  the  reaction  proceeds,  the  final  result  is  the  same.  But 
below  325°  this  is  not  the  case;  a  false  equilibrium  is  attained, 
which  varies  for  different  temperatures. 

These  considerations  apply  of  course  not  only  to  the  problem 
of  the  specificity  of  ferment  action,  but  to  the  general  proposition 
that  ferments  are  accelerations  of  existing  reactions,  and  not  re- 
actions de  novo. 

The  question  of  qualitative  specificity  of  ferments  has  a  nar- 
rower interest,  though  it  is  one  of  great  importance.  Do  the 
different  accelerators  of  a  reaction  yield  the  same  products,  or 
may  they  yield  different  products  ?  It  is  obvious  that  with  a 
pure  catalysor,  a  body  that  simply  diminishes  the  chemical  re- 
sistance of  a  substance,  the  nature  of  the  products  would  not  be 
altered.  But  in  the  frequent  atypical  fermentations,  where  the 
ferment  is  often  more  or  less  altered  in  the  course  of  the  reac- 
tion, some  alteration  in  the  products  might  be  expected.  Such 
alteration  might,  however,  not  be  properly  attributed  to  the  ca- 
talytic acceleration.  For  example,  a  certain  reaction  constitutes 
a  hydrolysis,  with  the  product  z.  The  ferment  enters  into  reac- 
tions with  z,  and  as  a  result  y  is  produced.  Here  we  have  a  new 
reaction,  one  not  connected'  with  the  acceleration,  but  one  that 
would  have  occurred  were  we  to  mix  z  and  the  ferment.  The  re- 
action constitutes  a  new  reaction,  a  secondary  reaction  in  the 
system.  An  illustration  of  this  is  seen  in  the  transformation  pre- 
viously described  of  cinchonin  into  the  isomeric  base,  a-i-cin- 
chonin  under  the  action  of  hydrochloric  acid.  The  product 
slowly  adds  hydrochloric  acid;  this  is  a  secondary  reaction  hav- 
ing no  relation  to  the  accelerated  transformation  of  the  cinchonin 
into  the  isomer,  but  simply  a  reaction  between  the  o-t-cinchonin 
and  HC1  such  as  would  occur  were  these  two  mixed  in  a  solution. 
A  further  possibility  lies  in  the  fact  that  the  products  may  react 
among  themselves  to  form  new  bodies,  which  are  of  course  not  to 


THE 

R 

OF 


VOL.  1]  Taylor. — On  Fermentation.  313 

be  classed  as  products  of  the  fermentation.  For  example,  argi- 
nase  accelerates  the  hydrolysis  of  arginine  to  urea  and  ornithin. 
The  fermentation  proceeds  well  at  a  slightly  alkaline  reaction. 
Now  the  alkali  accelerates  the  hydrolysis  of  urea  to  carbon  diox- 
ide and  ammonia.  The  appearance  of  ammonia  in  the  fermen- 
tation of  arginine  by  arginase  is  therefore  not  a  product  of  this 
fermentation.  We  must  thus  bear  in  mind  the  possibilities  of 
secondary  reactions — reactions  between  product  and  ferment,  be- 
tween product  and  solvent,  between  products  and  some  extrane- 
ous substance,  and  between  products  and  products.  An  excellent 
illustration  of  the  relations  to  an  extraneous  body  is  furnished 
by  the  Duclaux  experiments  on  the  chemical  fermentation  of  d- 
glucose.  When  the  sugar  is  exposed  to  sunlight  in  the  presence 
of  a  trace  of  sodium  hydroxide,  aethyl  alcohol  was  produced: 
when  calcium  hydroxide  was  employed  lactic  acid  was  produced. 
Now  since  the  accelerator  was  the  hydroxyl  ion,  which  was  the 
same  in  each  system,  the  difference  in  product  must  have  been 
due  to  some  action  of  the  calcium  as  against  the  sodium.  It  is 
clear  therefore  that  only  under  conditions  of  experimentation 
with  pure  substances  can  we  determine  whether  different  fer- 
ments produce  different  products.  Practically  all  the  studies  in 
connection  with  animal  ferments  may  be  regarded  as  worthless 
from  this  point  of  view.  For  example,  the  members  of  the  Hoff- 
meister  school  believe  that  the  ferment  connected  with  the  auto- 
lytic  digestion  of  the  liver  is  not  trypsin,  because  ammonia  is 
formed  in  the  auto-digestion,  while  it  is  not  found  in  ordinary 
tryptic  digestions.  Now  the  ways  by  which  ammonia  might  be 
produced  are  so  manifold  that  a  distinction  between  trypsin  and 
the  intracellular  ferment  based  solely  upon  that  finding  collapses 
of  its  own  instability.  The  glycerine  and  succinic  acid  that  occur 
in  alcoholic  fermentation  by  yeast  were  long  supposed  to  repre- 
sent integral  products  of  the  fermentation ;  we  now  know  that 
they  do  not  occur  in  the  fermentation  of  sugar  by  zymase,  and 
that  in  natural  fermentations  they  are  in  all  probability  not  de- 
rived from  the  sugar  at  all.  For  most  ferments  this  question 
cannot  be  discussed  at  all,  on  account  of  lack  of  data. 

Now  theoretically  a  ferment  may  be  conceded  the  power  of 
modifying  the  reaction  that  it  accelerates.     Wegscheider  in  his 


314  University  of  California  Publications.       [PATHOLOGY 

studies  on  catalysis  distinctly  reserved  such  a  group  of  catalyses, 
the  reactions  with  side  influences  as  he  expresses  it,  as  contrasted 
with  the  ordinary  accelerations,  reactions  with  only  direct  influ- 
ences. There  has  been  very  little  experimental  work  done  upon 
this  aspect  of  the  subject,  but  we  must  concede  the  theory  of 
Wegscheider.  A  further  possibility  for  qualitative  variations 
lies  in  the  fact  that  it  may  be  possible  for  a  reaction  to  follow 
more  than  one  equation,  and  these  may  be  accelerated  differently 
by  different  ferments.  Now  in  the  accurate  recent  studies  of 
fermentations,  with  the  use  of  purer  reagents  and  stricter  condi- 
tions, it  is  seen  that  the  distinctions  in  products  tend  to  disap- 
pear. Pepsin  we  now  know  yields  the  same  products  as  trypsin ; 
it  may  not  be  able  to  digest  all  the  same  proteins,  but  when  both 
digest  a  certain  protein,  the  same  products  are  formed.  All  the 
tendency  of  the  recent  study  in  the  fermentation  of  carbohy- 
drates has  been  in  the  direction  of  simplifying  the  products,  and 
we  may  confidently  expect  that  as  investigations  proceed  many 
of  the  deviations  in  products  commonly  held  to  be  specific  to  the 
ferment  will  disappear. 

On  the  contrary,  there  are  undoubted  instances  in  which  fer- 
ments modify  the  course  of  the  reaction  in  the  qualitative  sense. 
The  illustrations  that  at  once  come  to  mind  are  the  different  re- 
sults that  may  be  secured  with  fermentations  with  pure  cultures 
of  microorganisms  under  different  conditions.  Emmerling2  has 
only  recently  emphasized  the  statement,  based  upon  experimental 
work,  that  employing  a  constant  substrate,  pure  cultures  of  ;t 
germ  may  not  yield  the  same  products  under  different  external 
conditions  of  experimentation.  There  can  be  no  doubt  of  this. 
But  these  instances  cannot  be  directly  quoted  in  a  chemical  dis- 
cussion, because  of  the  complexity  of  the  relations.  We  have 
definite  chemical  illustrations.  The  hydrolysis  of  hydrolylamine 
in  alkaline  solution  follows  the  equation  :  3  NH3O  =  NH3  -f-  N2 
-f-  3  H20,  while  the  acceleration  of  the  reaction  with  platinum 
follows  the  equation  :  4  NH3O  =  2  NH3  +  N20  -f  3  H20.  The 
reactions  of  hydrazine  illustrate  the  modifications  that  may  be 
effected  by  other  relations  in  the  system.  Thus  in  simply  watery 
solution  the  reaction  with  platinum  runs  2  N,H4  =  2  NH3  -f-  N2 
-f-  H2,  wrhile  in  the  presence  of  an  alkali  the  reaction  is :  3  N2H4 


.  i]  Taylor. — On  Fermentation.  315 

=  2  NH3  -f  2  No  -f-  3  H2.  The  best  illustrations,  however,  have 
been  recorded  in  the  reversions,  and  these  are  of  great  value  be- 
cause  the  action  is  very  selective.  When  lactase  is  allowed  to  act 
upon  equal  parts  of  d-glucose  and  d-galactose  a  disaccharide  is 
formed;  this  is  not  lactose,  as  would  be  expected  since  the  syn- 
thesis is  the  ferment-reversion  of  lactose  cleavage;  the  sugar  is 
the  isomer  isolactose.  The  same  fact  holds  true  for  the  synthesis 
by  ferment  action  of  disaccharide  from  d-glucose  by  maltase ;  the 
sugar  is  not  maltose,  but  the  isomer  isomaltose.  These  modifica- 
tions cannot  be  explained  upon  any  other  basis  than  that  of  the 
qualitative  alteration  of  the  acceleration  by  the  ferment. 

This  question  then  resolves  itself  into  one  of  fact.  If  we  were 
to  find  two  ferments  that  give  in  general  the  same  products  of 
cleavage,  but  which  AVC  could  show  yielded  in  their  reversions 
different  substances,  we  should  be  warranted  in  classifying  them 
as  distinct.  Since  the  general  trend  of  fermentation  and  cataly- 
ses tends  to  identical  products,  we  should  not  reason  from  pro- 
ducts to  ferments  except  upon  the  basis  of  most  definite  experi- 
mental evidence.  Upon  the  other  hand,  we  must  recognize  that 
in  the  reactions  of  complex  organic  bodies  the  differences  in  chem- 
ical potential  between  the  original  body  and  the  products  are 
often  slight,  and  thus  several  possible  reactions  may  proceed  side 
by  side ;  thus  a  certain  ferment  might  accelerate  the  reaction  ac- 
cording to  one  equation  more  than  according  to  another  equation, 
and  a  different  ferment  might  exhibit  other  relations.  Therefore 
the  matter  resolves  itself  into  one  of  fact  solely. 


316  University  of  California  Publications.       [PATHOLOGY 


LITERATURE. 

Ciamician  and  Silber.    Berichte,  34,  1230,  2040-55,  1992,  3593,  4128-56,  1575- 

Condelli.    Gas.  Chim.  Ital.,  34. 

Cremer.     Zeitschr.  f .  Biol.,  32,  49. 

Dakin.     Jour.  Physiol.,  30,  253;  32,  199. 

Emmerling.1    Berichte,  32,  544. 

Emmerling.-    Die  Zersetzung  stickestoffreier  organ.  Substancen  d.  Bakterien, 

25ft. 
Fischer.1    Berichte,  27,  2036,  2985. 

Zeitschr.  f .  physiol.  Chem.,  26,  61. 

Berichte,  28,  1433. 

Fischer.2    Zeitschr.  f.  physiol.  Chem.,  26,  64. 
Fischer.3    Berichte,  35,  3144;  37,  3103. 

Zeitschr.  f.  physiol.  Chem.;  46,  52. 
Fischer.4    Zeitschr.  f.  physiol.  Chem.,  26,  83. 
Frankland  and  MacGreggor.     Chem.  News,  69,  33. 

Jour.  Chem.  Soc.,  63,  1028. 
Harden.     Chem.  Zeit.,  25,  353. 

van't  Hoff.    Vorles.  u.  theoret.  u.  physik.  Chem.,  1,  202. 
Lobry  de  Bruyn  and  van  Erenstein.     Berichte,  28,  3078,  3085. 

Eev.  Trav.  chim.  Pays-Bas.,  14,  116,  203-16,  262. 
McKenzie  and  Harden.    Jour.  Chem.  Soc.,  83,  1514. 
Markwald  and  McKenzie.    Berichte,  32,  2130;  34,  469. 

Proc.  Chem.  Soc.,  20,  41. 
Ost.     Zeitschr.  f.  angen.  Chem.,  18,  1170. 
Pelabon.    C.  r.  Acad.  Sc.,  124,  360. 
Piloty.     Berichte,  30,  3161. 
Pottevin.     Ann.  Pasteur.,  17,  31. 

38,  1176,  3813. 
Sachs.     Berichte,  37,  3425. 

Salkowski.     Zeitschr.  f.  physiol.  Chem.,  30,  478. 
Tollens  and  Schoene.  Chem.  Cenbl.,  69,  II,  967,  1012. 
Wegscheider.     Zeitschr.  f.  physik.  Chem.,  30,  593-5^,  290-55,  565. 
Wohl.     Berichte,  31,  1796. 


VOL.  1J  Taylor. — On  Fermentation.  317 


THE  K6LE  OF  FERMENTATION  IN  METABOLISM. 

THE  PROTEIN  METABOLISM. 

The  digestion  of  protein  is  an  act  of  fermentation  in  all  the 
essential  details.  Under  physiological  circumstances  the  protein 
is  not  entirely  converted  into  amido  acids ;  there  is  evidence  that 
a  certain  absorption  of  albumose  and  peptone  occurs.  That  the 
nitrogen  needs  of  the  body  may  be  maintained  upon  a  diet  of 
amido  acids,  the  completed  products  of  tryptic  digestion,  has 
been  clearly  shown.  The  act  of  absorption  is  apparently  an  act 
of  simple  diffusion,  though  this  is  not  clear  for  the  albumoses. 
The  conversion  of  the  products  of  digestion  into  the  proteins  of 
the  circulating  fluids  is  not  yet  generally  regarded  as  due  to 
ferment-action.  In  favor  of  the  theory  that  the  process  of  re- 
conversion is  the  reversed  action  of  the  proteolytic  ferments  is 
the  general  theory  of  the  catalytic  acceleration  of  reversed  reac- 
tions, and  the  concrete  demonstration  of  many  instances  of  such 
reversibility.  No  other  explanation  has  any  experimental  basis. 
Synthesis  by  cell  action  has  no  concrete  meaning,  and  is  in  any 
event  not  incompatible  with  the  idea  of  ferment  reversion.  That 
the  reversions  are  accomplished  with  such  great  rapidity  has 
been  admittedly  without  adequate  present  explanation  in  the  con- 
crete sense.  There  are,  however,  two  considerations  that  may  be 
adduced.  In  the  first  place,  there  may  be  conditions  resembling 
zymo-exciters.  Secondly,  it  must  be  recalled  that  the  conditions 
in  the  act  of  absorption  are  very  favorable  to  a  rapid  synthesis. 
In  the  act  of  absorption  we  have  the  products  of  digestion  dif- 
fusing through  a  colloidal  membrane,  a  semi-pernable  mem- 
brane, where  the  fluids  lie  in  closely  approximated  very  thin 
layers ;  in  short,  what  may  be  conceived  as  representing  an  ideal 
heterogeneous  system,  and  as  underlying  the  theory  of  Nernst, 
the  reactions  under  these  conditions  may  be  reasonably  assumed 
to  occur  with  great  rapidity.  The  cells  of  the  intestinal  mucosa 
contain  proteolytic  ferments  of  great  activity ;  we  have  no  reason 
to  believe  that  the  cells  purge  themselves  of  ferment  entirely ;  on 


318  Ciiit't  rsity  of  California  Publications.       [PATHOLOGY 

the  contrary,  it  would  be  natural  to  believe  that  the  colloidal 
meshes  of  the  cells  and  membrane  contain  ferment.  Now  the 
synthesis  is  known  to  be  complete  in  the  snbmucus  circulation; 
the  membrane  is  very  thin ;  the  fluids  of  circulation  perform  a 
constant  lavage.  Under  these  circumstances  the  colloidal  mem- 
brane in  which  the  reactions  are  occurring  is  very  thin,  and  thus 
the  factor  of  the  rate  of  diffusion  is  reduced  to  the  minimum, 
and  this,  with  the  postulated  great  rapidity  of  reaction  in  the 
heterogeneous  system,  might  adequately  explain  the  apparently 
disproportionately  rapid  rate  of  synthesis  of  protein  from  the 
products  of  digestion.  The  reconstruction  of  the  protein  mole- 
cule from  the  absorbed  products  of  digestion  is  usually  supposed 
to  occur  in  the  intestinal  mucosa.  The  portal  blood  is  known  to 
contain  more  amido  nitrogen  than  the  blood  of  the  general  cir- 
culation, and  it  is  possible  that  some  of  the  reconversion  is  done 
in  the  liver.  This  is  confirmed  to  some  extent,  in  an  indirect  way, 
by  the  fact  of  the  toxicity  of  the  portal  blood  when  introduced 
into  the  general  circulation  without  passage  through  the  liver,  as 
is  seen  in  the  Eck  fistula.  When  one  considers  the  lability  of 
proteins,  it  is  apparent  that  during  the  course  of  a  digestion  or 
of  the  subsequent  synthesis  abnormal  side  reactions  might  occur, 
and  these  might  conceivably  be  concerned  in  dyspepsia. 

The  reconstruction  of  the  protein  in  the  body  is  apparently 
not  simply  the  reformation  of  the  molecule  of  original  protein. 
The  biological  specificity  of  some  of  our  body  proteins  indicates 
that  qualitative  alterations  are  effected  in  addition.  Whether 
this  biological  specificity  is  bestowed  upon  the  molecule  of  pro- 
tein during  the  reconstruction  from  the  products  of  digestion  or 
after\vards  in  the  body  is  not  known.  The  fact  holds  for  serum 
globulin  rather  than  for  serum  albumin. 

It  is  not  yet  known  just  what  products  of  the  digestion  of 
protein  are  absorbed.  It  is  certain  that  products  are  formed  in 
the  stomach  that  do  not  give  the  biuret  reaction.  In  the  intestine 
many  amido  acids  are  known  to  occur.  There  is,  however,  a  frac- 
tion of  the  protein  molecule  that  seems  to  resist  the  digestion  with 
pepsin  and  trypsine,  a  polypeptide-like  substance,  that  may  be 
shown  on  acid  hydrolysis  to  contain  amido  acids.  Whether  erep- 
sinc  can  split  this  is  not  known.  Whether  this  could  be  absorbed 


VOL.  i]  Taylor.— On  Fermentation.  319 

is  not  known.  On  the  other  hand,  there  is  evidence  that  the  lower 
albumoses  and  peptone. may  he  absorbed.  There  is  no  demon- 
stration yet  that  in  natural  digestion  the  molecule. is  split  entirely 
to  the  simplest  amido  acids.  It  is  not  at  all  necessary,  whatever 
may  be  the  theory  of  the  origin  of  the  reconstructed  body  protein, 
to  assume  that  the  digestion  of  protein  must  carry  the  disinte- 
gration to  the  very  simplest  amido  acids.  It  is  easily  possible 
that  group  nuclei  may  be  condensible  to  protein.  An  illustration 
may  be  found  in  nuclein.  Here  the  group  nuclei  are  a  pentose, 
a  pyrimidine  body,  purin  bases,  and  phosphoric  acid.  There  is 
every  evidence  that  the  body  can  form  nuclein  from  these  group 
radicals ;  there  is  no  evidence  that  the  body  can  form  nuclein  from 
the  decomposition  products  of  these  groups.  Yet  this  is  what  is 
practically  postulated  in  the  assumption  that  protein  may  be 
formed  only  from  the  final  simple  amido  acids,  and  not  from 
groups  of  amido  acids.  The  process  of  reconstruction  may  be 
reasonably  divided  into  two  steps:  the  recombination  under  the 
influence  of  the  ferment,  and  the  reconstruction  of  the  molecule 
with  the  endowment  of  the  biological  properties  of  the  particular 
species.  Whether  this  be  done  in  one  or  two  stages  is  not  known. 
For  some  proteins  (serum  albumin)  it  seems  certain  that  there 
are  no  biological  specificities,  just  as  there  are  none  for  the  body 
sugar  and  fat.  It  might  be  conceived  that  the  simple  condensa- 
tion of  the  products  of  protein  digestion  occurred  in  the  intes- 
tinal mucosa  under  the  influence  of  the  proteolytic  ferment :  and 
that  the  biological  reconstruction  occurred  later  in  the  liver  and 
other  body  cells.  The  latter  might,  on  the  other  hand,  be  as  easily 
conceived  to  occur  in  the  intestinal  mucosa,  either  under  the  in- 
fluence of  the  reversing  ferment  (a  side  reaction),  or  of  other 
constituents  of  the  cell.  Whether  trypsine  or  erepsine  is  con- 
cerned in  the  reversion  is  not  known.  That  the  reversed  action  of 
a  ferment  may  lead  to  a  product  qualitatively  different  from  the 
original  substance  has  been  shown  for  maltase  and  lactase.  Mal- 
tase  accelerates  the  cleavage  of  maltose  into  d-glucose;  when  it 
accelerates  the  reversed  reaction,  the  product  is  not  maltose,  but 
isomaltose.  The  corresponding  fact  holds  for  lactase.  This  fur- 
nishes an  experimental  analogy  for  the  supposition  that  the  bio- 
logical stamp  is  placed  upon  a  body  protein  during  the  act  of 


320  University  of  California  Publications.       [PATHOLOGY 

synthesis  in  the  intestinal  mncosa.  It  is  a  current  misconception 
that  the  theory  of  the  reconstruction  of  the  protein  by  the  fer- 
ment action  must  imply  that  a  different  chemical  reaction  would 
occur  if  the  "building  stones"  (the  amido  acids)  were  condensed 
to  the  protein  by  the  action  of  the  body  cells.  The  essential  reac- 
tion in  either  case  is  the  same,  and  is  the  expression  of  the  chem- 
ical properties  of  the  amido  acids.  All  that  the  ferment  need  do 
is  to  accelerate  the  reaction,  by  lowering  the  internal  resistance 
to  the  reaction  resident  in  the  components.  It  might,  however,  in 
addition  introduce  a  side  reaction. 

Whatever  the  particular  proteins  in  the  diet — casein,  albumin, 
globuline,  vegetable  protein,  gelatine,  etc. — the  protein  formed 
from  them  as  the  result  of  digestion  is  apparently  the  blood  pro- 
tein alone ;  that  is,  serum  albumin  and  the  serum  globulins.  To 
this  statement  qualification  must,  however,  be  made  that  the  in- 
gested proteins  must  contain  sufficient  amounts  of  sulphur  and 
phenyl  groups  in  the  form  of  compound  amido  acids.  Thus  gela- 
tine cannot  cover  the  full  protein  need  of  the  body  because  it  is 
too  poor  in  phenyl  groups ;  but  combined  with  other  proteins,  it 
can  cover  half  of  the  protein  needs  of  the  body. 

The  different  proteins  seem  to  contain  nearly  all  the  same 
amido  acids.  The  quantitative  relations  are,  however,  very  dif- 
ferent with  the  different  proteins.  Under  the  circumstances,  the 
differences  in  proteins  (and  in  these  we  must  include  the  biolog- 
ical properties)  are  in  all  probability  to  be  regarded  as  the  ex- 
pressions of  different  intramolecular  arrangements  of  the  varying 
amounts  of  the  several  amido  acids.  Direct  analogies  for  this- 
view  are  to  be  noted  in  the  different  synthetic  peptides.  and  it  is 
indeed  what  should  have  been  naturally  expected  from  our  knowl- 
edge of  the  sugars  and  the  compound  benzol  substances.  That 
this  state  of  affairs  tends  to  favor  the  conception  of  the  asym- 
metry of  hydrogen  cannot  be  denied.  The  investigations  of  the 
Fischer  school  on  synthetic  peptides  are  of  the  most  fundamental 
importance  for  the  physiology  as  well  as  the  chemistry  of  protein. 

In  general  terms  therefore  we  may  speak  of  the  conversion  of 
these  several  proteins  into  the  blood  proteins  as  polymerizations, 
in  the  sense  that  the  amido  groupings  within  the  molecule  are  re- 
arranged. In  the  functions  of  the  body  now,  the  reverse  occurs:. 


VOL.  l]  Tdijlor.  —  On  F<  i  mentation.  321 


from  the  simple  blood  proteins  are  formed  the  different  proteins 
of  the  body  —  casein,  myogen,  gelatine,  reticuline,  etc.  Here  we 
have  the  specialization  of  particular  proteins,  again  by  the  pro- 
cess of  polymerization.  Now  polymerizations  under  such  circum- 
stances are  usually  autoreactions,  and  they  are  very  liable  to 
catalytic  accelerations.  Analagous  relations  hold  for  the  different 
hexoses,  as  will  be  pointed  out.  Theoretically  it  is  justified  to 
assume  that  these  reactions  of  protein  polymerization  within  the 
body  represent  fermentations. 

In  experiments  in  vitro,  we  learn  that  pepsin  and  trypsm  are 
both  able  to  accomplish  the  acceleration  of  the  hydrolysis  of  pro- 
tein to  such  an  extent  that  within  a  number  of  days  the  substrate 
is  in  large  part  converted  into  amido  acids.  Complete  the  reac- 
tion never  is.  Now  in  the  stomach  the  extent  of  peptic  digestion 
is  limited  by  the  very  short  duration  of  the  food  in  the  stomach, 
so  that  the  actual  .power  of  the  pepsin  does  not  become  apparent. 
Under  conditions  of  normal  digestion  little  amido  acid  is  formed 
in  the  stomach  :  indeed,  after  a  heavy  meal  all  of  the  protein  will 
not  be  converted  into  non-coagulable  protein,  and  at  no  time  is 
the  production  of  albumose  and  peptone  very  noteworthy.  This 
is  simply  a  matter  of  time,  for  if  the  ends  of  the  stomach  be 
ligated  and  the  contents  retained  for  a  number  of  hours,  it  will 
be  found  that  an  extensive  digestion  has  been  accomplished. 
Thus  we  learn  that,  although  the  secretory  activity  of  the  stom- 
ach could  perform  a  completed  digestion  if  accorded  sufficient 
time,  in  fact  the  chief  function  of  the  stomach  (apart  from  a 
possible  peptic  preparation  for  tryptic  and  ereptic  digestion) 
seems  to  be  to  mix  the  food.  and  discharge  it  gradually  into  the 
small  intestine.  This  knowledge  of  physiology  is  entirely  in  ac- 
cord with  the  general  experience  in  the  treatment  of  gastric  dis- 
ease. that  the  motility  of  the  stomach  is  its  most  important  func- 
tion. The  power  to  secrete  normally  is  an  invaluable  sign  of  the 
integrity  of  the  organ,  but  the  loss  of  the  secretory  power  with 
the  conservation  of  the  motor  power  may  be  of  little  injury  to 
the  individual,  while  the  loss  of  the  motor  function  usually  leads 
to  disturbances  of  grave  consequences.  The  actual  hydrolysis 
of  protein  is  in  practice  accomplished  largely  by  the  trypsin  and 
by  the  erepsin,  and  this  hydrolysis  is  far  in  excess  of  anything 


322  University  of  California  /'iihlications.      [PATHOLOGY 

we  can  accomplish  in  experiments  in  glass.  The  normal  diges- 
tion is  able  to  accomplish  within  a  few  hours  more  than  can  be 
accomplished  in  days  with  all  the  pancreatic  juice  secreted  in  a 
similar  space  of  time.  Bidder  and  Schmidt  have  shown  that  a 
cat  can  digest  and  assimilate  an  amount  of  protein  one-fifth  the 
body  weight  per  day;  but  with  one  day's  pancreatic  secretion  of 
a  cat  we  could  not  in  a  flask  begin  to  digest  so  much.  This  is  the 
same  experience  we  meet  with  every  fermentation ;  we  lack  in  the 
laboratory  experiment  some  condition  or  substance,  some  zymo- 
exciter,  that  multiplies  many  times  the  velocity  of  the  simple 
digestion  by  the  secretion  of  the  body.  Something  more  than 
enterokinase  or  tissue  substance  is  required  to  bring  the  velocity 
of  a  tryptic  digestion  in  glass  to  the  level  reached  in  intestinal 
digestion.  We  know  that  the  addition  of  succus  entericus  to  an 
active  pancreatic  juice  increases  its  activity,  and  this  we  must 
ascribe  to  the  presence  of  a  zymo-exciter.  The  regular  removal 
of  the  products  of  digestion  from  the  tract  by  absorption  would 
of  course  result  in  the  acceleration  of  the  reaction  velocity,  but 
this  factor  could  not  account  for  the  observed  differences  in  ve- 
locities in  artificial  and  natural  digestions. 

The  catabolisrn  of  protein  in  the  body  may  be  with  certainty 
regarded  as  fermentative.  For  this  statement  we  possess  three 
groups  of  evidence,  and  these  complete  the  chain.  We  know  in 
the  first  place  that  the  end-products  of  the  hydrolysis  of  protein 
are  various  amido  acids.  In  the  second  place,  it  is  possible  to 
isolate  intercellular  proteolytic  ferments.  Thirdly,  we  know  that 
the  formation  of  urea  may  be  traced  directly  to  the  amido  acids. 
That  amido  acids  are  the  end  products  of  the  protein  hydrolysis 
is  not  only  known  for  the  test  tube  experiment  and  then  applied 
to  the  cellular  functions ;  it  is  also  known  as  an  experimental  fact 
that  in  the  bacterial  and  autolytic  degenerations  of  organs  amido 
acids  are  formed,  and  that  under  conditions  of  severe  degenera- 
tion these  bodies  may  be  recovered  from  the  tissues,  blood,  and 
urine.  That  urea  is  formed  from  amido  acids  is  shown  by  the 
following  facts:  Many  amido  acids  when  ingested  are  elimi- 
nated as  urea ;  urea  may  indeed  be  formed  in  the  test  tube  by  the 
oxidation  of  amido  acids  in  the  presence  of  ammonia ;  when  the 
poly-amido  acid  arginine  is  hydrolyzed,  urea  is  formed.  It  is 


VOL.  l]  Taylor. — On  Fei-nif illation.  323 

further  known  that  ammonium  carbonate  when  ingested  or  in- 
jected into  the  portal  circulation  is  climated  as  urea,  and  that 
when  in  dogs  the  liver  is  switched  out  of  the  portal  circulation, 
ammonia  appears  in  the  urine  in  large  quantities.  The  urine  con- 
tains traces  of  amido  nitrogen,  and  yields  amido  acids  on  diges- 
tion with  acids.  This  represents  apparently  a  fraction  of  the  pro- 
ducts of  protein  hydrolysis  that  has  escaped  through  the  kidneys. 

The  reactions  whereby  urea  may  be  formed  from  amido  acids 
are  several.  Some  of  these  are  oxidations,  particularly  oxidative 
syntheses.  Of  these  an  illustration  may  be  given  in  the  forma- 
tion of  urea  from  oxaminic  acid.  Hoffmeister,  who  has  studied^ 
this  matter  most,  believes  that  the  adaptability  of  a  compound 
to  oxidation  to  urea  depends  upon  the  presence  of  the  groups 
rHXH2.COOH.  That  some  of  the  reactions  are  hydrolyses  is 
illustrated  by  the  formation  of  urea  from  arginine.  The  forma- 
tion of  urea  from  ammonia,  which  is  the  reversal  of  the  common 
fermentation  of  urea  into  ammonia  and  carbon  dioxide,  may  be 
regarded  simply  as  the  result  of  the  withdrawal  of  one  molecule 
of  water  from  the  ammonium  carbonate.  Dreschel  believed  that 
in  this  process,  as  well  as  in  the  formation  of  urea  from  the  amido 
acids,  the  reaction  passed  through  an  intermediary  stage  of  am- 
monium  carbamate,  so  that  under  this  interpretation  the  forma- 
tion of  urea  would  rest  upon  alternate  oxidation  and  reduction. 
For  all  of  these  theories  there  is  experimental  evidence,  but  we 
do  not. know  whether  one  or  all  actually  occur  in  the  formation 
of  urea  within  the  body. 

The  formation  of  the  urea  from  the  amido  acids  and  ammonia 
being  granted  as  a  chemical  procedure,  howr  do  we  know  that  the 
reaction  in  life  is  fermentative.  The  experimental  work  indi- 
cates that  at  ordinary  temperatures  and  concentrations  the  for- 
mation of  urea  from  amido  acids  and  ammonia  would  be  very 
slow,  while  in  life  it  is  rapid  and  capable  of  being  greatly  in- 
creased by  an  increase  in  the  protein  of  the  diet.  The  fresh  ex- 
tract of  liver,  as  first  described  by  Richet,  will  form  urea  from 
amido  acids.  The  fresh  extract  of  the  liver,  as  shown  by  Kossel, 
will  form  urea  from  arginine.  Confirmatory  evidence  may  be 
found  in  the  fact  that  several  of  the  synthetic  polypeptides,  con- 
densations of  amido  acids,  may  be  hydrolyzed  by  trypsin.  I  be- 


324  r ni versify  of  California  Publications.       [PATHOLOGY 

lieve  these  considerations  warrant  the  statement  that  the  forma- 
tion of  urea  in  the  animal  body  represents  a  fermentation. 

The  known  facts  for  the  metabolic  disintegration  of  protein 
may  be  summarized  as  follows.  The  protein  is  hydrolyzed  to 
amido  acids  by  the  intracellular  tryptic  ferment.  The  amido 
acids  may  be  then  split,  since  a  disamidation  ferment  is  known 
to  exist.  The  resulting  ammonia  is  then  available  for  the  forma- 
tion of  urea.  That  urea  is  not  formed  directly  from  amido  acids 
by  oxidation  is  not  denied;  that  it  is  formed  by  the  hydrolysis 
of  poly-amido  acids,  like  arginine,  is  quite  certain.  Thus  wp 

have: 

Protein  +  water  —  amido  acids. 

Amido  acid  +  water  =  fatty  acid  +  ammonia. 

Ammonia  -  >  ammonium  carbamate  -  >  ammonium  carbon- 
ate—>  urea.  It  is  possible  that  the  urea  and  ammonia  represent 
a  reversible  system  in  equilibrium  in  the  body ;  liver  extract  forms 
urea  from  ammonia  or  ammonia  from  urea  with  equal  facility. 

Creatinine  is  the  anhydride  of  creatine,  a  constant  constit- 
uent of  muscle.  It  is  in  all  regards  to  be  looked  upon  as  a  pro- 
duct of  the  metabolism  of  muscle,  and  bears  a  near  relationship 
to  urea,  into  which  it  may  be  converted  by  heating  with  alkalies. 
The  urinary  creatinine,  however,  seems  quite  independent  of  the 
urea  metabolism.  It  is  hydrolyzed  back  to  creatine  by  bacteria. 
The  relations  have  been  very  little  studied  for  these  reactions,  but 
there  can  be  little  doubt  that  what  has  been  said  of  the  urea  ap- 
plies by  analogy  to  the  creatinine,  and  that  they  are  fermentative. 

THE  PURIN  METABOLISM. 

The  purin  metabolism  is  independent  of  the  purin  input  in 
the  diet.  Ingested  purins  are  hydrolyzed  and  the  several  group 
constituents  are  absorbed.  Whether  the  absorbed  purin  bases 
are  utilized  in  the  purin  synthesis  is  not  known,  but  in  any  event 
the  purin  syntheses  of  the  body  are  entirely  independent  of  any 
purin  input.  The  synthesis  of  nuclein  comprises  the  combina- 
tion of  purin  bases,  pyrimidin  bodies,  pentose  sugar,  and  phos- 
phoric acid.  The  pentose  is  in  all  probability  derived  from 
hexose,  by  being  built  down  through  the  removal  of  one  atom  of 
carbon.  It  is  not  difficult  technically  to  build  a  sugar  down ;  this 


VOL.  l]  Taylor.—  On  Fermentation.  325 

is  accomplished  in  the  method  of  Wohl,  by  converting  the  hexose 
into  the  oxime,  which  is  transformed  into  the  corresponding  nit- 
rile,  from  which  one  atom  of  carbon  is  then  split  off  as  a  cyano- 
gen group  by  means  of  silver,  leaving  the  sugar  with  but  five 
atoms  of  carbon.  The  purin  bases  may  be  synthesized  from 
amido  acids,  or  from  urea  through  the  mediation  of  tartronic 
acid.  The  pyrimidin  derivatives  may  be  formed  from  protein, 
since  the  pyrimidin  ring  can  be  formed  from  either  the  amido  or 
the  guanidine  group.  Whether  these  syntheses  are  fermentative 
is  not  known.  The  great  importance  of  these  syntheses  has  been 
recently  emphasized  for  general  biology  by  Loeb,  who  has  indi- 
cated that  fundamental  problems  of  growth  and  development  are 
directly  related  to  the  synthesis  of  nuclein. 

Under  conditions  of  sterilization,  following  destruction  of  the 
tissue  ferments,  nuclein  is  slowly  hydrolyzed.  This  hydrolysis 
may  be  much  accelerated  by  increase  in  temperature,  by  acids, 
and  by  bacteria.  The  purin  catabolism  is  to  be  regarded  as  the 
enzymic  acceleration  of  this  hydrolysis.  Tissue  ferments  are 
known  that  hydrolyze  the  nuclein  to  the  component  purin  bases, 
the  pyrimidin  derivatives  (thymin,  uracile,  cytosin),  pentose.  and 
the  phosphoric  acid.  Our  knowledge  of  the  subsequent  fermen- 
tative relations  is  confined  to  the  purin  bases.  These  are  appar- 
ently adenine  and  guinine,  the  two  amido-purin  bases.  In  the 
acid  hydrolysis  of  nuclein,  the  purin  bases  yielded  are  adenine 
and  guanine  alone.  These  are  converted  into  hypoxanthin  and 
xanthin  by  the  disamidation  ferment  first  described  by  Jones. 
There  is  disagreement  between  the  different  investigators  whether 
the  ferments  that  convert  the  adenine  and  the  guanine  are  iden- 
tical ;  the  point  has  no  importance  for  the  theory  of  the  catabolism 
of  nuclein.  The  reactions  for  this  disamidation  are  as  follows  : 
Adenine  -f-  water  hypoxanthin.  —  ammonia. 

N=C.NH2  HN—  CO 

II  II 

HC     C—  NH  HC     C—  NH 


Guanine  +  water  xanthin  +  ammonia 

HN—  CO  HN—  CO 

II  II 

NH2.C     C—  NH  OC      C—  NH 

^PH  -I-  H  O  —  ^PW         -L-  1VTFT. 

N_C—  Nt=^°J  HN—  C—  N^C1  ^    •'• 


326  1'niro-Nity  of  Calif ornia  Publications.      [PATHOLOGY 

Tissues  contain  a  ferment  that  accelerated  the  oxidation  of 
the  hypoxanthin,  as  follows : 

Hypoxanthin  +  oxygen  =  xanthin. 
HN— CO  HN— CO 

HC     C— NH  +O=  OC     C— NH 

I  !l        \CH  !     II        \CH 

N— C— N=OJ  HN— C— N=ot 

Tissues  contain  a  ferment  that  accelerated  the  oxidation  of 
xanthin  to  uric  acid,  as  follows : 

Xanthin     -}-     oxygen  uric  acid 

HN— CO  HN— CO 

II  II 

OC     C— NH  OC     C— NH 

|          i!  NOT,  +0=  |          ||  \pn 

HN     C— N=0±  HN— C— NH^^ 

Following  this,  we  recognize  further  a  uricolytic  ferment  in 
tissues  that  are  active  in  the  cleavage  of  uric  acid ;  the  relations 
are  not  understood,  but  the  products  seem  to  be  urea  and  glyco- 
coll.  This  is  in  harmony  with  the  older  observations,  that  uric 
acid  is  converted  into  urea  in  the  body. 

Recent  investigations  tend  to  indicate  that  when  uric  acid 
is  oxidized  in  tissues  glyoxylic  acid  is  formed  as  an  intermediary 
product.  Since  the  final  product  is  urea,  the  reaction  may  be 
formulated  somewhat  as  follows:  (a)  Uric  acid  -|-  water  -f- 
oxygen  =  glyoxyl-urea  -)-  ammonia  -)-  carbon  dioxide ;  following 
which  the  glyoxyl-urea  would  be  oxidized  and  the  ammonia 
groups  united  with  the  carbonyl  groups  to  form  two  molecules  of 
urea.  (b)  Uric  acid  -f-  water  -|-  oxygen  =  allantoin  (glyoxyl- 
diureid),  which  would  on  hydrolysis  yield  urea  and  allanturic 
acid,  which  in  turn  would  be  hydrolyzed  to  urea  and  glyoxylic 
acid.  The  experiments  succeed  with  freshly  excised  or  perfused 
tissues. 

The  known  facts  indicating  that  the  catabolism  of  nuclein  is 
a  series  of  successive  fermentative  reactions  may  be  summarized 
as  follows : 

Nucleo-albumin  +  water  —  protein  +  nuclein. 

A  proteolytic  ferment. 

Nuclein  +  water  =  pentose,    pyrimidins,    ;miido-purins,    phosphoric 
acid. 

The  ferment  has  been  termed  a  nuclease. 


VOL.  i]  Ttii/Ior.—On  Fermentation.  327 

Amido  purins  +  water  =  purin  bases  +  ammonia. 
The  desamidation  ferment. 

Purin  bases  +  oxygen  =  uric  acid. 
An  oxydase. 

Uric  acid  -(-  oxygen  —  urea. 
Vricolytic  ferment. 

The  foregoing  considerations  apply  only  to  the  nucleinie  ca- 
tabolism.  Whether  this  is  the  sole  endogenous  source  of  the  purin 
bases  of  the  urine  is  not  known.  It  is  chemically  possible  that 
there  may  be  a  synthetic  formation  of  purin  bases,  independent 
of  the  oxidation  of  the  bases  derived  from  the  hydrolysis' of  the 
niiclein:  it  is,  however,  undemonstrated.  According  to  Burian, 
the  active  muscle  forms  purin  bases  (and  also  creatinine),  and 
this  cannot  be  derived  from  the  nuclein. 


THE   CARBOHYDRATE   METABOLISM. 

The  polysaccharides  of  the  diet  are  accelerated  in  their  hy- 
drolysis to  maltose  by  the  amylase  of  the  saliva,  the  pancreatic 
juice,  and  the  succus  entericus.  The  maltose  is  fermented  by  the 
maltase  of  the  saliva,  the  pancreatic  juice,  and  the  succus  enteri- 
cus. The  cane  sugar  is  fermented  by  the  invertase  of  the  saliva, 
the  pancreatic  juice,  and  the  succus  entericus ;  the  milk  sugar  is 
fermented  by  the  lactase  of  the  succus  entericus.  The  products — 
d-glucose,  d-laevulose,  and  d-galactose — are  absorbed  and  recon- 
densed  into  glycogen.  This  reconstruction  of  the  polysaccharide 
from  the  hexoses  we  may  assume  to  be  the  result  of  the  accelera- 
tion of  the  reversed  reaction  by  a  tissue  ferment,  a  glycogenase. 
-Whether  this  formation  of  glycogen  occurs  in  the  mucosa  of  the 
intestine  as  well  as  in  the  liver  is  not  known.  There  is  some  evi- 
dence that  it  occurs  also  in  the  muscles.  Between  d-glucose  and 
glycogen  a  relation  of  equilibrium  seems  to  exist,  again  an  expres- 
sion of  the  presence  of  a  ferment.  Whenever  the  glycogen  be- 
comes  excessive,  fat  is  formed.  The  fat  is  probably  formed  from 
the  sugar,  not  from  glycogen.  The  reaction  by  which  fat  is 
formed  from  sugar  is  not  known.  In  many  plants  the  reaction  is 


328  Cnircrsity  of  California  Publications.       [PATHOLOGY 

fermentative  and  reversible.  It  is  possible  that  a  disturbance  of 
the  d-glucose-glycogen  equilibrium  occurs  in  diabetes. 

When  glycogen  is  hydrolyzed,  only  d-glucose  is  obtained. 
Since  d-glucose,  d-fructose,  and  d-galactose  are  all  known  to  be 
formers  of  glycogen,  it  is  probable  that  before  the  hexose  is  con- 
densed to  the  polysaccharide,  the  d-fructose  and  the  d-galactose 
are  converted  into  d-glucose.  This  polymerization  may  be  easily 
accomplished  as  a  catalytic  reaction  by  the  action  of  hydroxyl 
ions,  and  apparently. this  polymerization  is  to  be  observed  in  the 
common  hydrolysis  of  starch.  Whether  this  conversion  of  the 
d-fructose  and  the  d-galactose  into  d-glueose  occurs  in  the  in- 
testinal wall  or  in  the  liver  is  not  known.  The  body  possesses  the 
power  of  reversing  this  reaction.  D-galactose  is  an  essential 
sugar  of  the  body,  being  present  not  only  in  the  milk,  but  also  in 
the  lipoids  of  the  central  nervous  system.  In  both  of  these  situa- 
tions it  is  formed  from  the  d-glucose  of  the  blood. 

When  sacchrose,  lactose,  and  maltose  are  injected  into  the 
circulation  they  are  eliminated  in  the  urine  quantitatively,  and 
this  indicates  that  the  blood  contains  no  invertase,  lactase,  or  mal- 
tase.  When  glycogen  is  formed  from  d-glucose,  we  have  the 
formation  of  a  polysaccharide.  Between  the  polysaccharide  and 
the  hexose  there  is  for  all  the  known  vegetable  starches  a  disac- 
charide  stage :  whether  this  exists  in  the  case  of  glycogen  is  not 
demonstrated. 

The  mechanism  of  the  combustion  of  sugar  is  not  known.  Ac- 
cording to  Cohnheim,  the  combustion  is  the  result  of  the  action 
of  two  substances :  one  contained  in  the  muscle,  the  other  in  the 
pancreas.  The  muscle  substance  is  the  accelerating  agent;  the 
pancreatic  substance  simply  activates  the  substance  in  the  muscle. 
Cohnheim 's  statement  has  been  contradicted,  and  we  must  await 
confirmation.  In  the  event  of  confirmation,  the  relation  of  the 
pancreatic  substance  would  be  either  that  of  an  activator,  like 
enterokinase,  or  that  of  an  zymo-exciter.  The  chemical  reaction 
of  the  combustion  of  sugar  is  unknown.  Stoklasa  has  described 
in  muscle  and  other  tissues  an  alcoholic  ferment,  and  suggests 
that  in  the  combustions  of  sugar  we  have  superimposed  an  alco- 
holic and  an  oxidation  fermentation,  the  sugar  passing  through 
the  stages  of  lactic  acid  and  alcohol  to  carbon  dioxide  and  water. 


"VOL.  1]  Tuijlor. — On  Fermentation. 

In  support  of  this  hypothesis  is  the  fact  that  traces  of  lactic  acid 
and  alcohol  are  to  be  found  in  all  fresh  tissues.  Muscle,  as  Her- 
mann demonstrated,  will  produce  carbon  dioxide  in  an  atmos- 
phere free  of  oxygen.  The  experimental  findings  that  in  the 
autolysis  of  muscle  the  alcohol  is  not  increased,  though  sugar  is 
destroyed  and  carbon  dioxide  produced,  does  not  speak  against 
the  Stoklasa  scheme,  since  the  alcohol,  being  but  an  interme- 
diary stage,  would  be  expected  to  be  present  only  in  momentary 
traces.  What  makes  one  pause  in  the  acceptance  of  the  hypothe- 
sis are  the  magnitudes  concerned.  If  the  entire  combustion  of 
sugar  be  supposed  to  pass  through  the  stage  of  alcohol,  that 
means  that  for  the  average  body  each  kilo  of  active  tissue  (ex- 
cluding the  fat  and  skeleton)  would  need  to  burn  0.010  g.  of 
alcohol  per  minute.  Of  course  in  such  a  reaction  the  alcohol 
would  exhibit  only  a  momentary  transitory  appearance;  never- 
theless the  quantities  concerned  are  such  as  will  make  one  hesi- 
tate. The  hypothesis  requires  confirmation,  but  if  it  can  be 
confirmed,  a  long  step  in  advance  will  have  been  made.  Whatever 
the  reaction  of  combustion,  there  can  be  no  question  that  it  is  a 
fermentative  accelerative. 

On  the  assumption  that  the  combustion  of  sugar  passes 
through  the  stage  of  aethyl  alcohol,  Stoklasa  and  Bach  suggest 
the  following  scheme  of  progression : 

glucose  =  lactic  acid. 

CH,.OH  (<JHOH)4COH  =  2  CH3.CH (OH) .COOH. 

lactic  acid  =  aethyl  alcohol  -f  carbon  dioxide. 

2  cHa.CH(OH).COOH  =  2  CH3.CH2.OH  +  CO2. 

aethyl  alcohol  +  oxygen  =  acetic  acid  +  water. 

2  CH3.CH,.OH  +  02=  2  CH3.COOH  +  2  H2O. 

acetic  acid  —  methane  -f  carbon  dioxide. 

i'  ( '  1 1  ,.<  'OOH  =  2  CH4.  +  2  CO,. 

Methane  +  oxygen  =  formic  acid  +  water. 

2  ( 'H4  +  3  O,  =  2  H.COOH  +  2  H2O. 

formic  acid  +  oxygen  =  carbon  dioxide  +  water. 

•1  H.COOH  +  2  O.,  =  2  CO2  +  2  H2O. 

This  scheme  is  to  some  extent  possibly  corroborated  by  the  fact 
that  in  the  early  stages  of  an  alcoholic  fermentation  the  produc- 
tion of  alcohol  is  relatively  greater  than  that  of  carbon  dioxide. 
Should  this  scheme  be  confirmed  it  will  afford  an  opportunity  to 


330  University  of  California  Publications.       [PATHOLOGY 

determine  at  just  what  point  if  any  below  the  stage  of  sugar  the 
combustion  in  the  diabetic  is  impaired. 


THE  FAT   METABOLISM. 

The  digestion  of  fat  is  the  simplest  reaction  of  hydrolysis. 
The  absorption  of  the  products,  however,  is  less  well  understood. 
Physiologists  have  for  years  been  resolved  into  two  camps  with 
regard  to  the  digestion  and  absorption  of  fats.  Bernard  first 
observed  the  emulsification  produced  by  the  action  of  the  pan,- 
creatic  juice,  and  this  state  of  emulsification  has  been  long  re- 
garded as  an  important  fact  in  the  theory  of  fat  absorption. 
This  emulsion  is  due  but  in  part  to  the  presence  and  action  of 
the  pancreatic  juice ;  it  depends  in  part  upon  the  soaps  and  upon 
the  bile.  On  the  other  hand,  evidence  has  been  accumulating 
tending  to  show  that  the  hydrolysis  of  the  fat  is  a  necessary  con- 
dition preparatory  to  absorption.  The  direct  proof  of  the  syn- 
thesis of  fat  is  afforded  by  the  experiment  of  injecting  free  fatty 
acid  and  glycerine;  they  are  absorbed  as  fat,  and  this  combina- 
tion may  be  effected  by  the  resected  intestinal  mucosa.  Within 
recent  years  the  facts  have  become  so  clear  that  we  are  now  war- 
ranted in  reaching  a  conclusion.  This  conclusion,  to  which  the 
work  of  Pflueger  in  late  years  has  contributed  much,  is  that  all 
fats  are  hydrolyzed  before  absorption,  and  that  they  are  not  ab- 
sorbed at  all  as  finely  divided  particles  in  the  state  of  emulsifi- 
cation. The  microscopic  pictures  that  were  formerly  relied  upon 
by  the  Haidenhain  school  to  demonstrate  the  direct  absorption  of 
fat  particles  are  seen  on  careful  investigation  to  be  easily  capable 
of  different  interpretation.  Furthermore,  many  instances  have 
been  adduced  in  which  finely  divided  bodies  in  a  state  of  emulsi- 
fication, as  paraffine,  have  been  introduced  into  the  intestine,  but 
never  absorbed.  On  the  other  hand,  the  work  of  Rosenfeld  and 
others  has  shown  that  when  a  peculiar  fat  is  introduced  into  the 
intestine  and  absorbed,  that  particular  fat  is  to  be  found  chemi- 
cally intact  in  the  flesh  of  the  animal.  Thus  linseed  oil  and  eru- 
caic  oil  are  to  be  found  in  the  liver  following  their  ingestion, 
and  it  is  possible  to  feed  a  starved  dog  with  mutton  solely  and 
have  firm  mutton  fat  deposited  all  over  the  dog,  conferring  upon 


v«>""i]  Taylor.— On  Fermentation.  331 

the  animal  a  hardness  to  the  touch  very  different  from  the  nor- 
mal condition.  The  total  evidence  upon  fat  metabolism  and  de- 
position leads  to  the  postulation  of  two  rules  that  may  be  accepted 
as  well  established.  When  animals  construct  their  fats  from  their 
natural  diet,  in  the  case  of  herbivora  from  carbohydrates,  the  fat 
is  specific  to  the  species.  Thus  horses  and  cattle  on  the  same  diet 
of  grasses  and  grain  will  construct  different  fats.  When,  on  the 
other  hand,  animals  invest  fats,  that  fat  is  deposited  in  the  tissues 
unaltered.  Thus  the  fat  of  a  dog-  may  be  made  to  consist  solely 
of  mutton  fat.  In  a  word,  synthesized  fat  is  specific  to  the 
species,  absorbed  fat  to  the  diet.  Since  this  is  true,  it  is  apparent 
that  upon  the  view  that  fats  are  absorbed  not  directly  but  only 
after  cleavage,  we  must  assume  that  after  the  fats  are  hydro- 
lyzed,  in  the  act  of  absorption  (after  passing  the  [for  fats]  non- 
permeable  membrane),  they  are  again  recombined  into  their  orig- 
inal states.  This  .is  the  only  interpretation  that  will  correspond 
to  the  facts,  and  do  justice  to  the  theory  that  fats  are  only  ab- 
sorbed after  cleavage.  Xow  we  know  of  but  one  way  in  which 
this  combination  of  the  fatty  acid  and  glycerine  may  be  accom- 
plished, and  that  is  by  a  reversion  of  the  reaction  accelerated  by 
the  fat-splitting  ferment.  With  the  probability  of  this  proposi- 
tion we  are  entirely  satisfied,  since  instances  of  fat  synthesis 
through  the  reversed  action  of  lipase  have  been  demonstrated. 
But  the  details  are  not  at  all  clear.  This  combination  of  the  fatty 
acid  and  glycerine  must  be  conceded  to  be  effected  with  great 
rapidity,  since  large  quantities  of  fat  may  be  absorbed  within 
a  few  hours  and  the  contents  of  the  thoracic  duct  contain  no  free 
fatty  acid.  It  is  therefore  necessary  to  postulate  the  presence  of 
some  condition  very  favorable  to  the  reversed  action  of  the  fer- 
ment, such  as  a  zymo-exciter.  We  are,  however,  lacking  in  in- 
formation of  a  chemical  nature  bearing  upon  such  a  condition. 
There  is  also  the  further  difficulty  that  we  are  not  able  to  under- 
stand the  rapidity  with  which  the  fatty  acids  must  pass  the  mu- 
cous membrane.  The  higher  fatty  acids  diffuse  very  slowly,  yet 
in  actual  digestions  large  quantities  pass  through  the  intestinal 
wall  within  a  few  hours.  Naturally  one  considers  the  possibility 
of  combinations  of  the  fatty  aoids  with  other  substances,  combi- 
nations possessing  a  greater  rate  of  diffusion.  But  what  sub- 


332  University  of  California,  Publications.       [PATHOLOGY 

stances?  Not  soaps,  for  they  diffuse  practically  not  at  all,  as 
Krafft  has  shown.  Pflueger  has  indicated  combinations  with 
some  of  the  constituents  of  the  bile,  combinations  subject  to  a 
high  degree  of  hydrolytic  dissociation.  But  such  combinations 
have  not  been  isolated  and  shown  to  possess  rapid  rates  of  diffu- 
sion. Pflueger  laid  great  stress  upon  the  conditions  that  work 
for  the  free  solubility  of  the  fatty  acids  and  a  rapid  cleavage, 
but  that  does  not  entirely  elucidate  the  problem  of  absorption, 
since  this  is  an  act  of  diffusion,  and  solubility  and  diffusibility 
do  not  necessarily  go  hand  in  hand.  Granted  therefore  that  fats 
are  absorbed  only  after  hydrolysis,  and  that  on  absorption  they 
are  recoinbined  through  the  agency  of  the  reversed  activity  of 
lipase,  the  rapidity  of  absorption  of  the  fatty  acids  and  to  some 
extent  the  rapidity  of  the  fat  synthesis  are  not  understood.  One 
cannot  forbear  the  suggestion  that  possibly  the  lipoidal  contents 
of  the  cells  of  the  intestine,  the  lecethins,  etc.,  may  play  a  promi- 
nent role  in  these  transactions,  but  the  suggestion  is  a  purely 
hypothetical  one.  Nevertheless  it  seems  that  the  relations  of  the 
Nernst  hypothesis  previously  alluded  to  find  direct  application  to 
the  intestinal  mucosa. 

Not  only  do  we  not  understand  the  rapidity  of  the  absorption 
of  the  hydrolyzed  fats  through  the  intestinal,  we  do  not  fully 
understand  the  velocity  of  intestinal  hydrolysis.  One  of  the  most 
striking  facts  in  fat  fermentations  is  the  slowness  of  cleavage  with 
lipases  in  vitro  as  compared  with  the  velocity  of  the  same  reac- 
tion in  vivo.  This  is  not  true  of  lipase  alone;  it  seems  to  hold 
good  for  all  the  animal  ferments,  and  for  many  of  those  of  vege- 
table origin.  It  has  thus  been  long  obvious  to  the  students  of 
fermentation  that  some  condition  must  be  present  in  the  body 
that  is  not  duplicated  in  the  experiment.  For  pancreatic  lipase 
we  have  recently  been  instructed  as  to  the  nature  of  one  favor- 
able condition  through  the  observation  of  Hewlitt  that  lecethin 
acts  as  a  powerful  zymo-exciter  for  the  ferment.  The  accelerat- 
ing action  of  bile  upon  the  lypolytic  function  of  the  pancreatic 
secretion  has  been  long  known,  but  never  before  elucidated. 
Hewlitt  has  determined  that  a  trace  of  lecethin  will  accelerate 
notably  the  cleavage  of  fats  by  the  pancreatic  secretion.  This 
influence  he  has  shown  cannot  be  due  to  the  conversion  of  a 


VOL.  i]  Taylor. — On  Fermentation.  333 

zymogen  into  active  ferment,  nor  to  any  alteration  in  the  reac- 
tion ;  nor  is  it  an  additive  function,  since  the  lecethin  alone  is  in- 
active. Probably  some  component  of  lecethin  operates  as  well 
as  the  lecethin,  since  the  action  persists  after  the  heating  of  the 
lecethin  to  199°,  a  temperature  at  which  this  substance  is  rapidly 
disintegrated.  Bile  contains  an  appreciable  quantity  of  lecethin, 
and  until  the  contrary  is  demonstrated,  we  are  justified  in  ascrib- 
ing the  accelerating  action  of  the  bile  on  pancreatic  lypolysis  to 
the  zymo-exciting  action  of  lecethin.  As  illustrating  the  specific 
relations  that  are  encountered  in  these  matters,  I  may  say  that 
lecethin  has  no  influence  upon  the  cleavage  of  fat  by  the  ferment 
of  the  castor  bean.  A  somewhat  analogous  observation  for  the 
lipase  of  the  liver  is  that  reported  by  Magnus.  When  the  solu- 
tion of  lipase  was  dyalized  it  lost  its  activity,  though  this  was  at 
once  restored  when  a  little  boiled  extract  of  liver  was  added ;  i.e., 
a  necessary  cooperating  substance  was  removed  by  the  dyalysis. 
The  substance  could  be  roughly  isolated ;  it  was  precipitable  by 
uranium  acetate  in  the  presence  of  protein,  also  by  basic  lead 
acetate,  insoluble  in  alcohol  and  aether,  and  was  destroyed  by 
ashing.  Obviously  the  extract  of  the  liver  contains  a  lipase  and 
a  zymo-exciter,  or,  as  Magnus  calls  it,  following  the  nomenclature 
of  Bertrand,  a  co-ferment. 

The  conception  of  a  fat  hydrolysis  as  stated  for  the  insoluble 
higher  fats  may  then  be  applied  to  the  digestion  of  fat  within 
the  intestine,  with,  however,  several  modifications.  In  the  first 
place,  alkali  is  present  to  some  extent,  and  during  some  time 
(though  both  have  been  largely  overrated),  and  thus  some  of  the 
fatty  acids  (the  products)  are  combined  and  for  the  purposes  of 
the  relations  in  the  system  removed.  This  would  tend  to  reduce 
the  concentration  of  products,  and  to  that  extent  would  facili- 
tate the  rate  of  transformation.  Secondly,  fatty  acid  is  being 
constantly  removed  by  resorption,  and  this  would  further  re- 
duce the  concentration  of  fatty  acid  in  the  system.  We  observe 
therefore  that  even  under  the  interpretation  of  the  velocity  of  a 
fat-hydrolysis  being  simply  a  velocity  of  diffusion,  the  removal 
of  the  fatty  acid  (and  the  glycerine  also,  it  being  resorbed) 
would  tend  to  accelerate  the  progress  of  the  transformation.  It 
is  at  the  same  time  clear  that  were  we  to  consider  that  the  reac- 


M4  University  Of  California  I'llhl leal  ions.        [PATHOLOGY 

tions  in  the  zone  of  contact  of  the  two  phases  were  not  practically 
instantaneous,  but  to  some  extent  dependent  upon  the  active  mass 
of  the  substrate  and  the  products,  the  removal  of  the  products 
would  accelerate  greatly  the  velocity  of  the  transformation. 
There  is  without  doubt  some  practical  bearing  in  these  consid- 
erations. In  the  problems  of  every  disturbance  in  fat  digestion, 
particularly  in  infants  and  children,  one  has  to  consider  the  ab- 
sorption from  the  intestine  as  well  as  the  hydrolysis  on  the  part 
of  the  lipase  of  the  pancreatic  juice.  Now  there  are  instances 
where,  with  obviously  faulty  absorption  of  the  fats,  there  is  ap-, 
parently  some  reduction  in  the  total  hydrolysis  of  the  fats,  al- 
though there  may  be  good  evidence  that  the  pancreatic  secretions 
are  performing  normally  their  proteolytic  and  amylytic  func- 
tions. For  these  cases  the  nearest  explanation  lies  directly  in 
line  with  the  facts  just  alh4pd  to;  the  lagging  in  the  fat  cleavage 
iii,-iy  be  looked  upon  as  the  result  of  the  non-removal  of  the  fatty 
acids  by  absorption,  the  acceleration  resulting  from  the  removal 
of  the  products  is  wanting,  and  we  have  in  the  child's  intestine 
simply  the  velocity  of  the  system  in  a  closed  chamber.  It  is 
therefore  very  probable  that  even  with  an  intact  functionation 
of  the  pancreas,  the  rate  of  cleavage  of  the  fats  of  a  normal  diet 
might  be  so  reduced  as  to  become  clinically  appreciable,  simply 
because  of  defective  resorption  of  the  fatty  acids.  The  distur- 
bances in  fat  digestion  in  children  are  probably  in  the  large  ma- 
jority of  cases  dependent  upon  faulty  absorption,  due  to  atrophic 
enteritis,  amyloid  deposition  in  the  intestine,  tuberculosis  of  inu- 
cosa  and  retroperioneal  lymph  glands,  tuberculous  peritonitis, 
lymphatic  hyperplasis  of  the  retroperitoneal  and  mysenteric 
glands  of  unknown  origin — and  not  to  any  disturbances  in  the 
pancreatic  functions.  In  such  cases  a  certain  reduction  in  the 
cleavage  is  to  be  expected,  and  the  most  likely  explanation  is  the 
one  just  stated. 

Of  the  combustion  of  fats  we  possess  still  less  definite  informa- 
tion. Whether  the  fats  are  burned  directly  or  first  converted 
into  sugars  is  an  old  question  that  has  not  been  decided.  In  the 
.-vent  of  a  direct  utilization  of  the  fat,  its  combustion  would  com- 
prise the  two  reactions  of  hydrolysis  and  oxidation.  We  are  not 
in  a  position  to  speculate  upon  the  mechanism  of  the  reaction. 


VOL.  i]  Taylor. — On  Fermentation.  335 

The  fatty  acids  of  the  series  CnH2n02  may  be  regarded  as  pro- 
ducts representing  the  successive  additions  of  the  group  CH2  to 
the  general  construction  H.COOH,  which  is  the  formula  for  the 
lowest  member  of  the  group,  formic  acid.  Now  the  direct  oxida- 
tion of  formic  acid  may  be  easily  shown  to  follow  the  reaction : 
2  H.COOH  +  0  =  COOH.COOH  +  H2O.COOH.COOH  +  0  = 
H20  -(-  2  C02,  oxalic  acid  being  the  intermediary  product.  There 
is  no  theoretical  reason  why  the  oxidation  of  the  higher  fatty 
acids  should  not  follow  a  similar  course,  the  groups  CH2  being 
successively  split  off  under  oxidation  to  water  and  carbon  diox- 
ide. We  know  by  experience  that  acetic,  formic  and  oxalic  acids 
are  formed  in  oxidations  of  the  higher  fatty  acids.  We  have, 
however,  no  direct  knowledge  that  these  or  similar  reactions  oc- 
cur in  the  body ;  it  would  not  be  difficult  to  represent  the  forma- 
tion of  the  final  products  by  other  equations.  Thus  the  fatty 
acid  might  be  oxidized  directly,  through  the  oxyacid;  and  an 
intramolecular  cleavage  would  be  equally  feasible  from  the  chem- 
ical point  of  view.  The  possibility  that  the  fats  are  first  con- 
verted into  sugars  has  long  been  assumed,  though  despite  numer- 
ous attempts  it  has  not  been  experimentally  demonstrated  in  a 
conclusive  manner.  There  can  be  no  doubt  of  the  existence  of 
the  opposite  reaction,  the  conversion  of  sugar  into  fat.  Though 
the  steps  of  this  conversion  are  not  known,  beyond  the  fact  that 
fatty  acids  are  very  commonly  formed  in  the  disintegrations  of 
sugars,  the  daily  experience  of  the  fat-forming  power  of  sugar 
demonstrates  the  occurrence  of  the  process.  And  it  is  quite  nat- 
ural therefore  to  assume  that  the  contrary  reaction,  the  formation 
of  sugar  from  fats,  may  be  a  common  physiological  occurrence, 
and  this  assumption  is  supported  by  the  known  occurrence  of 
this  reversible  reaction  in  the  vegetable  organism.  Gautier  has 
described  these  reactions  as  processes  of  fermentation,  and  has 
suggested  equations.  Now  while  it  is  apparent  that  the  conver- 
sion of  sugar  into  fat  and  the  reconversion  of  fat  into  sugar  may 
be  acts  of  fermentation  and  from  the  point  of  view  of  general 
physiology  probably  are  to  be  classed  as  acts  of  fermentation,  we 
possess  absolutely  no  chemical  data  tending  to  the  concrete  ex- 
perimental demonstration  of  the  thesis.  The  combustion  of  these 
bodies  for  the  maintenance  of  the  body  heat  must,  however,  be 


336  University  of  California,  Publications.       [PATHOLOGY 

believed  to  be  of  fermentative  nature ;  the  wide  variations  in  ve- 
locity, without  variations  in  concentration  and  temperature,  alone 
compel  us  to  the  designation  of  fermentation. 

The  fats  of  the  body  are  concerned  in  very  important  syn- 
theses, the  transformations  into  the  lipoidal  bodies,  including  the 
various  lecethins  and  the  complex  fatty  substances  of  the  central 
nervous  system.  These  transformations  are  in  general  additions 
of  fatty  moieties  to  other  complex  molecules ;  of  the  nature  of  the 
reactions  we  know  nothing.  So  far  as  we  know, 'these  syntheses 
are  very  slow  processes,  solely  connected  with  growth,  reproduc- 
tion of  cells  and  regeneration,  in  all  probability  syntheses  of  slow 
velocity  and'  not  subject  to  fluctuations.  The  lipoidal  bodies  are 
of  the  greatest  physiological  importance,  and  doubtless  constitute 
bodies  of  the  highest  dignity.  The  little  we  know  of  them  does 
not  indicate  that  their  formation  represent  fermentations. 

Fats  circulate  in  a  soluble  and  dialyzable  state,  apparently  in 
the  form  of  some  complex  combination.  This  fact,  as  stated  by 
Connstein,  is  easy  of  confirmation.  The  facts  suggest  that  this 
soluble  and  dialyzable  state  represents  the  active  state  of  fat  in 
the  mass  reaction  sense,  while  neutral  fat  is  but  the  storage  state. 


Facts  are  also  present  in  the  physiology  of  the  secretion  of 
the  digestive  juices  that  suggest  strongly  fermentative  accelera- 
tions. As  Bayliss  and  Starling  have  shown,  the  secretion  of  the 
pancreatic  juice  is  stimulated  by  the  action  of  a  substance  formed 
in  the  intestinal  mucosa,  which  they  term  secretin,  and  which  acts 
by  a  direct  influence  upon  the  cells  of  the  pancreas,  to  which  it 
is  carried  by  the  circulation.  Histological  studies  had  previously 
suggested  that  the  pancreatic  cells  form  and  store  in  their  proto- 
plasm granules  from  which  the  zymogen  is  formed,  termed  there- 
fore prozyrnogen.  Apparently  the  secretin  accelerates  the  con- 
version of  the  prozymogen  into  zymogen,  which  is  then  secreted. 
Whatever  may  be  the  actual  relations,  the  process  suggests 
strongly  a  fermentative  acceleration.  The  activation  of  the 
zymogens  into  the  active  ferments  likewise  appears  to  be  of  en- 
zymic  nature.  Bayliss  and  Starling  have  demonstrated  for  the 
activation  of  trypszymogen  by  enterokinase  that  the  reaction  is 


VoL- 1]  Taylor.— On  Fermentation.  337 

one  of  hydrolysis,  that  it  follows  the  law  of  mass  action,  and  that 
the  velocity  of  activation  is  proportional  to  the  mass  of  the  ente- 
rokinase.  Similar  relations  may  be  assumed  to  hold  for  the  pro- 
cesses of  activation  of  the  other  zymogens. 

Another  interesting  reaction  in  the  secretion  of  the  alimentary 
juices  is  to  be  found  in  the  secretion  of  hydrochloric  acid.  Just 
what  the  mechanism  of  this  reaction  is  we  do  not  know.  But 
since  the  concentration  of  the  available  components  of  the  several 
possible  equations  for  the  formation  of  hydrochloric  acid  remains 
constant,  and  the  temperature  is  constant,  the  exaggerated  for- 
mation of  hydrochloric  acid  during  digestion  must  represent  a 
simple  act  of  positive  catalysis.  Of  experimental  evidence  we 
have  none.  But  how  else  is  one  to  interpret  the  enormous  varia- 
tions in  the  velocities  of  these  transformations  and  secretions,  if 
they  be  not  deemed  fermentative? 


In  a  discussion  of  the  relations  of  ferment  action  to  the  pro- 
cesses of  disease,  we  possess  as  yet  more  uncultivated  than  culti- 
vated territory.  In  few  lines  are  the  relations  sufficiently  simple 
and  the  data  uncontradicted  to  enable  us  to  define  in  even  gen- 
eral terms  the  scope  that  may  be  properly  allotted  to  fermenta- 
tions. 

In  a  great  many  diseases  we  have  coagulation  necroses  occu- 
pying a  most  prominent  position  among  the  lesions.  Thus  early 
in  diphtheria,  pneumonia,  tuberculosis  and  many  other  infectious 
conditions  we  meet  with  coagulations  as  essential  features  of  the 
lesions.  The  coagulation  itself  does  not  seem  in  any  essential 

manner  different  from  the  coagulation  of  the  blood  plasma.  Now 
the  definition  of  coagulation  as  a  fermentation  rests  simply  upon 

the  occurrence  of  the  phenomenon  in  a  rapid  manner,  without 
dependence  upon  alterations  in  concentration  of  reacting  bodies 
or  temperature,  and  upon  the  lack  of  any  proportional  relation 
between  the  quantity  of  the  active  agent  and  the  extent  of  the 
transformation.  That  these  coagulations  occur  slowly  in  the  ab- 
sence of  the  accelerator  we  do  not  know.  Milk  has  been  preserved 
sterile  and  neutral  for  years,  without  any  curdling  having  de- 
veloped. Thus  we  judge  these  phenomena  as  being  fermentative 


338  University  of  California,  Publications.       [PATHOLOGY 

without  the  occurrence  of  the  reaction  or  process  in  the  original 
system  being  known  or  even  assumed.  Granted,  however,  that  co- 
agulation at  low  temperature  as  the  result  of  the  addition  of  some 
cellular  extract  is  an  act  of  fermentation,  these  pathological  co- 
agulations have  the  same  rank.  These  coagulations  occur  with 
great  velocity,  and  may  affect  tissues  in  a  widespread  fashion. 
Since  many  of  these  coagulations  occur  in  connection  with  bac- 
terial infections,  the  assumption  is  usually  made  that  the  coagu- 
lating ferment  is  derived  from  the  bacteria  directly,  or  at  least 
derived  from  the  reaction  between  the  germs  and  the  tissues.  We 
are,  however,  acquainted  with  instances  in  which  the  action  of 
chemical  substances  results  in  coagulation  necrosis  at  the  site  of 
application. 

Liquefactions  also  occur  in  the  body  under  conditions  that 
point  strongly  to  their  definitions  as  fermentations.  So  far  as 
we  can  observe,  these  liquefactions  (decoagulations)  differ  in  no 
known  point  from  the  first  stage  of  protein  digestion.  Some  in- 
sults to  tissues  result  directly  in  liquefaction,  but  it  is  much  more 
common  for  it  to  be  preceded  by  coagulation.  The  coagulation 
necroses  are  usually  followed  by  liquefaction,  and  it  is  through 
this  agency  that  the  removal  of  the  coagulated  material  is  effected. 
Not  only  does  this  apply  to  coagulated  protein,  it  applies  to  pro- 
cesses of  necrobiosis.  Whenever  cells  involved  in  a  pathological 
lesion  undergo  processes  of  disintegration,  we  have  good  reasons 
for  the  belief  that  they  are  first  digested  and  then  the  soluble 
products  removed.  The  absorption  of  pus  is  a  good  illustration. 
Pus  does  not  usually  resorb,  but  this  is  due  rather  to  the  contin- 
uation of  the  activity  of  the  pyogenic  agent  than  to  the  resistance 
of  the  pus  itself  to  digestion.  Now  under  some  circumstances, 
when  the  pyogenic  agent  is  weak  or  dies  out,  the  pus  is  slowly 
liquefied  and  the  products  gradually  resorbed.  This  occurs  some- 
times in  the  peritoneal  and  pleura!  cavities,  in  both  localities  in 
some  instances  involving  large  masses  of  pus.  Some  purulent 
pleural  effusions,  those  due  to  the  pneumococcus  in  particular, 
show  a  very  slight  virulence,  and  will  undergo  complete  resorp- 
tion,  even  though  the  quantity  of  pus  be  large.  The  pus  cells 
certainly  do  wander  back  into  the  circulation  of  their  own  ac- 
count. It  wrere  conceivable  that  leucocytes  could  invade  the  pus 


VOL.  1]  Taylor. — On  Fermentation.  339 

cavity  and. return  to  the  general  circulation  bearing  an  amorph- 
ous load,  and  the  occurrence  of  this  cannot  be  denied.  But  the 
study  of  these  purulent  exudates  yields  so  many  chemical  signs 
of  protein  hydrolysis  that  we  are  driven  to  the  conclusion  that 
the  chief  process  lies  in  the  digestion  of  the  dead  cells  in  situ,  and 
the  removal  of  the  soluble  products  of  this  digestion  by  resorp- 
tion.  The  exudate  in  crupous  pneumonia  is  removed  in  the  same 
manner.  Very  little  of  the  consolidated  exudate  is  removed  by 
expectoration ;  the  mass  is  liquefied  and  digested  and  the  pro- 
ducts removed  by  resorption.  Direct  experiments  with  the  pneu- 
monic lung  have  demonstrated  a  rapid  rate  of  digestion  under 
these  conditions.  The  same  fact  has  been  demonstrated  for  the 
uterus  in  involution  and  for  the  degenerated  liver.  We  are,  I 
think,  justified  in  the  general  assumption  that  necrobiotic  cells 
are  disposed  of  by  means  of  a  veritable  digestion.  Furthermore, 
it  is  known  that  in  the  columns  of  the  spinal  cord  suffering  from 
sclerosis  the  quantity  of  myelin  and  lecethins  is  greatly  reduced, 
consequences  best  interpreted  as  the  result  of  digestion.  In  the 
acute  fat  necrosis  of  the  pancreas  we  have  an  instance  of  a  direct 
exhibition  of  the  action  of  a  ferment.  The  marked  disintegration 
of  the  pancreas  in  many  cases  of  acute  pancreatitis  may  in  a  sim- 
ilar manner  be  best  explained  as  the  result  of  the  action  of  the 
trypsin  upon  the  pancreas  itself. 

Similar  findings  are  encountered  in  the  degenerations  that  are 
produced  as  the  results  of  poisonings  with  known  chemical  sub- 
stances. In  the  degenerations  of  the  liver  that  result  from  the 
action  of  phosphorus  and  to  a  less  extent  arsenic,  we  find  in  the 
presence  in  the  liver  and  blood  of  noteworthy  quantities  of  amido 
acids,  direct  evidence  that  digestion  processes  have  been  active. 
Not  only  this,  but  the  direct  experiment  with  the  self-digestion 
of  the  phosphorus  liver  reveals  a  marked  increase  in  the  rate  of 
self -digestion  as  compared  with  the  normal  liver.  In  acute  yel- 
low atrophy  and  the  analogous  conditions,  we  have  in  all  proba- 
bility complete  analogues  of  these  toxic  degenerations  of  the  liver. 

Tumors  sometimes  undergo  regressive  processes  with  lique- 
factions that  resemble  in  all  respects  these  digestions  of  organs. 
In  many  instances  these  processes  of  reversions  in  tumors  are 


340  University  of  California  Publications.       [PATHOLOGY 

undoubtedly  the  result  of  bacterial  infection,  but  in  some  in- 
stances the  results  cannot  be  so  interpreted. 

Now  it  is  not  a  characteristic  of  all  conditions  of  necrobiosis 
and  coagulation  necrosis  that  the  removal  of  the  material  is  ac- 
complished through  the  agency  of  liquefaction  and  digestion.  In 
particular  is  this  not  the  case  in  tuberculosis.  The  detritus  con- 
tained in  a  cold  abcess,  erroneously  termed  pus,  is  extremely  re- 
sistant to  digestion.  Though  the  contents  of  these  cold  abcesses 
are  fluid,  they  incline  to  persist  for  years;  the  amorphous  par- 
ticles and  the  suspended  colloids  resist  digestion  and  the  persist- 
ence of  these  exudates  is  often  a  serious  condition.  The  same 
thing  is  true  of  the  caseous  material  in  pulmonary  tuberculosis ; 
it  may  be  encysted,  encapsulated,  and  even  encalcified,  but  the 
material  exhibits  the  greatest  integrity.  On  chemical  analysis, 
the  material  exhibits  an  analogous  behavior.  It  is  extremely  re- 
sistant to  solution  in  all  media,  and  extremely  resistant  to  diges- 
tions with  known  ferments.  In  short,  the  caseous  material  con- 
ducts itself  in  the  test  tube  just  as  it  does  in  the  body.  That  it  is 
a  protein  is  known  largely  through  the  content  of  nitrogen,  and 
through  the  results  of  putrefaction  and  of  acid  hydrolysis,  to 
which  the  material  is,  however,  extremely  resistant. 

We  have  in  the  general  occurrence  of  ferments  in  tissues  the 
best  reason  for  invoking  them  in  the  explanation  of  these  phe- 
nomena. It  is  now  an  experimentally  demonstrated  fact  that 
autohydrolysis  occurs  whenever  fats,  carbohydrates,  and  proteins 
are  kept  in  pure  water  under  aseptic  conditions.  These  have  been 
long  known  for  the  starches,  the  disaccharides  and  the  esters,  in- 
cluding the  fats.  I  have  during  the  past  two  years  demonstrated 
this  autohydrolysis  for  casein,  globuline,  nuecleo-albumine,  pro- 
tainine,  and  gelatine.  These  materials  behave  in  this  manner  not 
only  in  the  isolated  state  but  also  when  they  are  integral  parts 
of  tissues.  All  fresh,  undiseased,  sterile  organs  will  on  standing 
exhibit  auto-digestion,  and  the  autolyses  affect  not  only  the  pro- 
tein but  likewise  the  nuclein,  the  fat,  and  the  carbohydrates. 
The  velocity  of  these  transformations  is  very  slow;  it  is  only 
under  conditions  of  infection,  inflammation,  and  necrobiosis  that 
the  velocity  of  these  transformations  is  increased.  Whether  such 
accelerations  be  due  to  the  formation  of  greater  quantities  of  fer- 


VOL.  l]  Taylor. — On  F<  run  illation.  341 

inent  or  also  to  some  alteration  in  attendant  conditions  (in  mass 
relations)  we  do  not  know.  It  is  also  possible  or  probable  that 
among  the  products  of  bacterial  metabolism  are  bodies,  different 
from  their  specific  poisons,  that  act  as  accelerators. 

While  the  general  effects  of  bacteria  upon  tissues — the  com- 
mon infective  lesions  of  the  cells — are  in  all  probability  not 
specific  to  the  microorganism,  since  they  occur  in  all  infections 
to  some  degree  arid  may  reasonably  be  assumed  to  be  of  the  na- 
ture of  fermentations;  the  specific  intoxications  are  not  to  be  so 
explained.  The  toxines  of  bacteria  are  specific  to  the  microor- 
ganism, and  not  to  the  host.  They  are  in  all  probability  produced 
to  some  extent  in  whatever  medium  of  life  the  germ  is  stationed, 
in  the  body  of  a  susceptible  animal  or  upon  an  artificial  culture 
medium.  The  formation  of  the  toxine  within  the  microorganism 
may  in  all  theoretic  possibility  represent  an  act  of  fermentation. 
Its  action  upon  .the  host  cannot  be  so  explained.  This  we  must 
simply  -class  with  the  specific  actions  of  poisons  like  atropine, 
hydrocyanic  acids,  carbon  disulphide,  ricine,  and  mercury,  as  a 
reaction  de  novo.  There  is,  ho\vever,  one  general  distinction  be- 
tween the  bacterial  toxines  and  the  usual  vegetable  and  mineral 
poisons;  under  favorable  conditions  the  host  will  develop  anti- 
toxines,  whereas  anti-bodies  to  the  other  poisons  are  not  devel- 
oped. To  this  statement  there  are  exceptions.  Some  bacteria, 
under  the  present  conditions  of  experimentation  at  least,  do  not 
provoke  the  formation  of  anti-toxines,  while  a  few  vegetable  pois- 
ons, like  ricine.  do  provoke  the  formation  of  anti-bodies.  The  fact 
that  all  the  known  substances  that  provoke  anti-bodies  are  colloids 
is  in  itself  of  little  direct  importance.  Of  the  formation  of  these 
anti-toxines  we  have  absolutely  no  chemical  knowledge  or  expla- 
nation. There  is  a  vast  amount  of  data  bearing  upon  the  physical 
and  chemical  relations  of  toxines  to  anti-toxines,  into  which  order 
has  been  introduced  through  the  quantitative  measurements  and 
calculations  of  Arrhenius  and  Madsen.  Upon  the  question  of  the 
modus  operandi  of  the  formation  of  the  anti-toxine  after  the  in- 
jection of  the  toxine  we  possess  no  chemical  or  physical  data. 
The  hypotheses  of  Ehrlich  have  thus  far  not  been  productive  of 
chemical  or  physical  results. 


OF   THE 

UNIVERSITY 

OF 


[ERRATUM. — On  page  300;  fifth  line  from  bottom  of  page,  instead  of  CH2 
group  read  CH  —  group.] 


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Lfifoirairy 


JANgp 


APR  2  5  1960 
IN  BIOLOGY  STtACKS 

APR  1 2  1950 


RECEIVED 


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CIRCULATION 


BY 

1987 

OEPt. 


LD  21-50m-l,'3 


Taylor,  A*E 
On  ferInellta^ 

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